Formulations for mediating inflammatory bowel disorders

ABSTRACT

The invention provides formulations and methods for mediating inflammation, in particular an inflammatory bowel disorder such as necrotizing enterocolitis, and for. Further, the formulations are effective in lowering blood cholesterol and decreasing blood cholesterol absorption. The formulations comprise at least one ganglioside, which may be selected from the group consisting of: GD3, GM1, GM2, GM3, and GD1b. The invention provides a method of treating or preventing inflammatory diseases, such as necrotizing enterocolitis by delivery of at least one ganglioside to a subject in need thereof. Supplementation of foods or liquids with gangliosides, fore example infant formula or infant foods, can be employed according to the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of, claims priority from, andis entitled to the full benefit of U.S. patent application Ser. No.10/551,789 which entered U.S. national phase on Sep. 30, 2005, and whichis the national phase of International Patent ApplicationPCT/CA2004/000375 filed Mar. 12, 2004. This application also claimspriority from and is entitled to the full benefit of U.S. patentapplication Ser. No. 10/404,095 filed Apr. 2, 2003. Each of theapplications noted above is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to a formulation for mediatinginflammation, such as inflammatory bowel disorders.

BACKGROUND OF THE INVENTION

Gangliosides are specialized sialic acid-containing glycolipids abundantin the outer region of the neuronal lipid bilayer and intestinal brushborder. The intestine contains a relatively high amount of ganglioside(as much as 7% of total lipids (Christiansen et al, 1981; Forstner etal., 1973). Change occurs in the composition and molecular structure ofgangliosides during intestinal development (Bouhours et al., 1983;Glickman et al, 1976). Total intestinal lipids are comprised of 25 to35% sphingolipids, including gangliosides and sphingomyelin(Christiansen et al, 1981; Forstner et al., 1973), and microvillusmembranes are more enriched in gangliosides than the plasma membranes(Forstner et al., 1973).

Gangliosides are located at the surface of the cell membrane with thehydrophilic oligosaccharide chain extending into the extracellularspace. Glycosphingolipid constitutes approximately 20% of the brushborder membrane lipids (Forstner et al., 1973). The dominant gangliosideis GM3 which is 7 times more concentrated in the neonatal compared toadult intestine of rats (Bouhours et al., 1983). The specificphysiological roles of gangliosides are poorly understood, however,studies showed that gangliosides provide binding sites for a wide rangeof pathogens including viruses, bacteria and fungi (Holmgren et al.,1985; Kyogashima et al., 1989; Laegreid and Otnaess, 1987; and Rolsma etal., 1998). For example, ganglioside GM3 acts as a natural receptor inpig small intestine for rotavirus (Rolsma et al., 1998) and theenterotoxigenic bacteria Escherichia coli (E. coli) K99 (Kyogashima etal., 1989). Ganglioside GM1 in human intestine (Holmgren et al., 1985)and in human milk (Laegreid et al., 1987) also provides receptors forenterotoxin of Vibrio cholerae and the heat-labile E. coli, therebyacting as a physiological barrier for protection against these entericinfections.

Previous studies showed that gangliosides exist in clusters in theplasma membrane forming glycosphingolipid enriched domains and thatthese domains are the preferential interaction sites between targetcells and pathogens (Karlsson, 1995). Preterm newborn infants fedganglioside supplemented formula at a concentration of 1.43 mg/100 Kcal,were shown to have significantly lower numbers of E. coli andbifidobacteria in the feces (Rueda et al., 1998).

During early development, important morphological changes occur in thetotal and relative amounts of gangliosides in neuronal tissues of thebrain and retina (Asou et al. ,1989; Baumann et al., 1976; Daniotti etal., 1994). One of the primary roles of gangliosides is activation ofneuronal cell differentiation and proliferation (Ledeen et al., 1998),influencing synaptogenesis and neuritogenesis (Byrne et al., 1983;Svennerholm et al., 1989) and offering protection against neuronalinjury (Guelman et al., 2000; Mohand-Said et al., 1997). Functions inthe intestinal mucosa involve toxin receptors of bacterial and viruses(Thompson et al., 1998; Rolsma et al., 1998) and immune activators(Vazquez et al., 2001). Radiolabeling studies have shown that exogenousgangliosides and sphingomyelin are hydrolyzed by enterocytemembrane-bound enzymes such as sphingomyelinase and/or ceramidase(Merrill et al.,1997; Schmelz et al., 1994; Nilsson, 1968). Metabolitessuch as ceramide, ceramide-1-phosphate, sphingosine, andsphingosine-1-phosphate are transported into enterocytes and reutilizedin synthesis of gangliosides or sphingomyelin or both (Merrill etal.,1997; Schmelz et al., 1994). Since gangliosides and sphingomyelinare incorporated into lipoproteins and chylomicrons (Hara et al., 1987;Merrill et al., 1995), dietary gangliosides, sphingomyelin and/or theirintestinal metabolites are likely to be transported throughout the bodyto affect sphingolipid biosynthesis in other organs (Vesper et al.,1999). Studies have suggested a possible interaction betweensphingolipids and phospholipids (Merrill et al.,1997; Schmelz et al.,1994; Ogura et al., 1988). Sphingosine-1-phosphate is metabolized intoethanolamine phosphate and hexadecanal, both prerequisite materials forphospholipid synthesis (Merrill et al.,1997; Schmelz et al., 1994).Radiolabeled ganglioside [³H]sphingosine-GM1 when injectedintraperitoneally into mice was incorporated into hepatocytephospholipids in the EPL form (Ogura et al., 1988).

Ether Phospholipids. To date, there has been no research investigatingwhether dietary gangliosides can be used for synthesis of EPL in theintestine or influence EPL synthesis in neuronal tissues. EPL have anester linkage at the sn-2 position, but have an ether linkage, either toan alkyl or alkenyl group, at the sn-1 position. EPL tend to be enrichedin mammalian intestinal and neuronal cells (Paltauf, 1972). One type ofEPL known as plasmalogens (a group of1-O-alkenyl-2-acyl-glycero-phospholipids), accounts for about 75% ofethanolamine phosphoglycerides (EPG) in myelin of rat brain, 65% of EPGin human brain (Horrocks, 1972) and 12% of EPG in rat intestinal mucosa(Paltauf, 1972). High content of EPL may contribute to maintenance ofcell integrity and function (Alonso et al., 1997; Bittman et al., 1984;Diomede et al., 1993; Honma et al., 1981; Mavromoustakos et al., 2001;Oishi et al., 1988; Paltauf, 1994; Principe et al., 1994; Seewald etal., 1990; and Zheng et al., 1990). EPL can affect membrane propertiessuch as permeability (Bittman et al., 1984) and fluidity (Paltauf,1994). EPL influence signal transduction to many metabolic pathways byprotein kinase C (PKC) (Zheng et al., 1990), Na-K-ATPase (Oishi et al.,1988), inositol-lipid turnover (Seewald et al., 1990), and intracellularcalcium (Alonso et al., 1997). EPL induce cell apoptosis (Alonso et al.,1997), cytotoxicity (Diomede et al., 1993; Honma et al., 1981), andantitumor activity (Mavromoustakos et al., 2001; Principe et al., 1994),which could have potential in anti-cancer applications. Selectivecytotoxic effects of EPL is dependent on membrane cholesterol amount(Diomede et al., 1990). For example, HL60 cells with a high cholesterolcontent show lower uptake of EPL into membranes, resulting in decreasein membrane fluidity (Diomede et al., 1990) and higher rates ofapoptosis (Diomede et al., 1993). Alkyl-lysophospholipids exhibit strongselective cytotoxicity in leukemia cells but not in normal bone marrowcells (Honma et al., 1981).

Gangliosides and EPL may perform similar functions. For example, both ofthese types of lipids localize in neuronal (Byrne et al., 1983;Svennerholm et al., 1989; and Horrocks 1972) and intestinal tissues(Forstner et al., 1973; Paltauf, 1972). Both gangliosides and EPLexhibit anti-cancer effects (Mavromoustakos et al., 2001; Principe etal., 1994; Schmelz et al., 2000) and contribute to cell differentiation(Ledeen et al., 1998; Honma et al., 1981) and apoptosis (Diomede et al.,1993; Malisan et al., 2002). Gangliosides and EPL are sensitive tomembrane cholesterol content (Diomede et al., 1990; Blank et al., 1992).

In American diets daily intake of SPL (including gangliosides andsphingomyelin) is about 300 mg (Vesper et al., 1999) and daily intake ofEPL is about 1 mg per gram of food (Berger et al., 2000). Neonatesconsume SPL and EPL from mothers milk (Diomede et al., 1991), but themetabolic interaction between dietary SPL and EPL is not known.

Cholesterol reduction in membranes causes increased EPL uptake (Diomedeet al.,1990; Leikin et al., 1988) and increased activity of Δ-5 and Δ-6desaturase enzymes (Clandinin et al., 1991). Dietary gangliosides mayincrease total membrane EPL content and accompanied with higherpolyunsaturated fatty acid (PUFA) in subclasses of EPL. Sphingomyelincan be used as a control to compare bioavailability with gangliosidesbecause sphingomyelin and gangliosides have the same ceramide moleculeanchored in the cell membrane, but attached to a different head group.Using rats, the present data illustrated herein demonstrates thatdietary ganglioside increases total content and composition of EPLcontaining PUFA in the developing intestine.

Microdomains. Microdomains, generally called lipid rafts, caveolae, orglycosphingolipid-signaling domains, have been characterized asimportant domains for signal transduction and lipid (i.e. cholesterol)and protein trafficking (Anderson, 1998; Brown et al., 1998; Hakomori etal., 2000; and Simons et al. 1997). Microdomains are recently known as asite for the cellular entry of bacterial and viral pathogens (Fantini,2000; Katagiriet al., 1999; and Bavari et al., 2002). For instance, theentry of filoviruses requires lipid rafts as the site of virus attack(Bavari et al., 2002). Cholera toxin entered the cell by endocytosis GM1as the sorting motif necessary for retrograde trafficking into hostcells and such trafficking depends on association with lipid rafts (Wolfet al., 2002).

Physiological and functional roles of microdomains are dependent oncholesterol and sphingolipids including gangliosides. Reduction ofcholesterol inhibits pathogen entry by disrupting the structure ofmicrodomains (Popik et al., 2002; Samuel et al., 2001) and impairsinflammatory signalling (Wolf et al., 2002; Triantafilou et al., 2002).Cholesterol upregulates the expression of caveolin, a marker of proteinfor caveolae (Fielding et al., 1997; Hailstones et al., 1998).Sphingolipid depletion inhibits the intracellular trafficking ofGPI-anchored proteins and endocytosis via GPI-anchored proteins(Kasahara et al., 1999), suggesting that lipid-protein interactiondirectly modulates gene expression and cellular trafficking importantfor cell development and behaviour.

The neonatal intestine has permeable, endocytic and enzymatic transportsystems for absorption of nutrients and immunoglobulins (Moxey et al.,1979; Wilson et al., 1991) but is susceptible to pathogen entry becauseof higher permeability than that of adults (Koldovsky 1994). High amountof gangliosides in mothers' milk during the neonatal period thereforeact as a receptor for viral and bacterial toxins to protect entry ofpathogens into enterocytes (Rueda et al., 1998). During development,membrane permeability gradually decreases (Koldovsky 1994) whilepeptidases and glycosidases become functionally active and enriched inmicrodomains (Danielsen et al., 1995). Many digestive/absorptiveenzymes, such as alkaline phosphatase, aminopeptidase N and A, andsucrase-isomaltase are also increased in apical membrane microdomains(Stulnig et al., 2001). These results seem to suggest the importance ofmicrodomains of intestinal apical membranes for nutrient uptake andmetabolism.

Polyunsaturated fatty acids (20:5n-3 or 22:6n-3) can accumulate inmicrodomains and displace functional proteins by changing the lipidcomposition of the microdomain (Stulnig et al., 2001; Williams et al.,1999). This observation highlights the importance of dietary lipids inmodulating physiological and biological properties of proteins in themicrodomain. Little is known of how dietary gangliosides affect thelipid profile and protein components of microdomains during neonatal gutdevelopment.

Some previous studies have suggested that cholesterol depletion inhibitsinflammatory signaling by disrupting microdomains structure (Wolf etal., 2002; Samuel et al., 2001; Triantafilou et al., 2002). However, ithas not been evaluated whether diet-induced cholesterol reduction hasany effect on decreasing cholesterol in the microdomain, disruptingmicrodomain structure and reducing pro-inflammatory mediators such asdiglyceride (DG) and platelet activating factor (PAF). DG derived fromphospholipids by phospholipase C, binds to protein kinase C (PKC) tophosphorylate targeted proteins, such as the epidermal growth factorreceptor and DG resides in microdomains (Sciorra et al., 1999; Smart etal., 1995). The instant invention assesses these effects.

PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine, stimulatesinflammatory cells such as leukocytes (Prescott et al., 1990) andactivates phospholipase A2 (PLA2) in the intestinal tissue to releasearachidonic acid (Okayasu et al., 1987). Meanwhile, increased lyso-PC byPLA2 is further used for PAF synthesis with an acetylcholinetransferase. PAF binds its receptor to increase intracellular calciumand inositol triphosphate (IP3) production and PKC activation forinflammation (Flickinger et al., 1999). It is unknown if PAF alsolocalizes in the microdomain. Since several studies reported thatsphingomyelin (SM), a sphingolipid, has an inhibitory effect on PLA2activity (Koumanov et al., 1997), it was of interest to determine ifdietary ganglioside also decreases PAF synthesis either by increasingsphingolipids or by disrupting microdomains structure in developingintestine. We also examine if dietary ganglioside reduces DG content inthe microdomain since sphingosine, a derivative of sphingolipidsinhibits PKC signaling which is required a structural complex with DG.

Neonates consume SPL including gangliosides from mothers milk (Carlson1985; Berger et al., 2000). Gangliosides are known to act as receptorsfor viruses and toxins (Laegreid et al., 1987; Rolsma et al., 1998),activators for T-cells (Ortaldo et al., 1996) and stimulators for Th-1and Th-2 cytokine-secreting lymphocytes in neonates (Vazquez et al.,2001). Gangliosides are also one of the major lipid components inmicrodomains. It is not known if dietary ganglioside changes the lipidprofile and structure of the intestinal microdomain and modulatinginflammatory signalling mechanisms in the developing intestine.

Necrotizing enterocolitis is an inflammatory bowel disease of neonatesand remains the leading gastrointestinal emergency in premature infants.Since current treatments disrupt the natural microflora, are invasiveand do not target the specific processes underlying development ofnecrotizing enterocolitis, it remains the leading cause of morbidity andmortality in neonatal intensive care units. Prematurity,hypoxia-ischemia, infection and formula feeding are established riskfactors, and breastfeeding is protective.

It is desirable to find a compound, a class of compounds, or compositionactive in mediating inflammation. It is also desirable to find suchcompounds or compositions that is naturally occurring in the foodsupply, so as to more easily meet with public acceptance.

Further, it is desirable to find a compound, a class of compounds, orcomposition active in mediating inflammation. Advantageously, suchcompounds or compositions would be naturally occurring in the foodsupply, so as to more easily meet with public acceptance.

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Abbreviations used herein are as follows:

CPG: Choline phosphoglycerides; DG: diacylglycerol; E. Coli: Escherichiacoli; EPG: Ethanolamine phosphoglycerides; EPL: Ether phospholipids;Gang-High: High concentration of ganglioside; Gang-Low: Lowconcentration of ganglioside; GD1b: II3 (NeuAc)₂-GgOse₄Cer; GD3:Ganglioside GD3:II³ (NeuAc)₂-LacCer; GLC: Gas liquid chromatography; GG:gangliosides; GM1: Ganglioside GM1:II³ NeuAc-GgOse₄Cer; GM2: GangliosideGM2:II³ NeuAc-GgOse₃Cer; GM3: Ganglioside GM3:II³ NeuAc-LacCer; LCPUFA:Long chain polyunsaturated fatty acids; LPC: lysophosphatidylcholine;LPE:lysophosphatidylethanolamine; MUFA: monounsaturated fatty acids;NANA: N-Acetyl neuraminic acid; PAT: platelet activating factor; PBS:Phosphate buffered saline solution; PC:phosphatidylcholine; PE:phosphatidylethanoloamine; PI: phosphatidylinositol; PKC: Protein kinaseC; PL, phospholipids; PS: phosphatidylserine; PUFA: polyunsaturatedfatty acids; SEM: Standard error of the mean; SFA: Saturated fattyacids; sIgA: Secretory immunoglobulin A; SM: sphingomyelin; SPL:Sphingolipids; TG: Triglyceride; and TLC: Thin layer chromatography.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a formulation,including a compound, a class of compounds or a composition active inthe mediation of inflammatory bowel disorders. Such mediation ofinflammation can decrease cholesterol absorption.

According to embodiments of the invention, a formulation is providedthat is effective in mediating inflammation. The formulation comprisesat least one ganglioside in combination with an acceptable excipient formediating an inflammatory bowel disorder, which may more particularlymean preventing or treating an inflammatory bowel disorder, such asnecrotizing enterocolitis in particular.

The ganglioside may selected from the group consisting of: GD3, GM1,GM2, GM3, and GD1b. The formulation may be in the form of a supplementedliquid or food, such as an infant formula or infant food.

The ganglioside may comprise about 80% GD3, 9% GD1b, and 5% GM3 on aweight/weight basis, or one of the gangliosides in the formulation maycomprises more than 50% of ganglioside content.

A method is provided for mediating an inflammatory bowel disorder in asubject in need thereof comprising the step of administering at leastone ganglioside to said subject. Administering may comprise oralconsumption. The inflammatory bowel disorder may be necrotizingenterocolitis.

A method is provided for reducing blood cholesterol in a subject in needthereof comprising the step of providing at least one ganglioside tosaid subject for oral consumption. The ganglioside may be selected fromthe group consisting of: GD3, GM 1, GM2, GM3, and GD1b.

Other aspects and features of the present invention will become apparentto those ordinarily skilled in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described, by way ofexample only, with reference to the attached Figures, wherein:

FIG. 1 illustrates the ratio of SFA to MUFA (white columns) and SFA toPUFA (black columns) in alkenylacyl-, alkylacyl- and diacyl subclassesin CPG in intestinal mucosa of animals fed control or treatment diets.

FIG. 2 illustrates the ratio of SFA to MUFA (white columns) and SFA toPUFA (black columns) in alkenylacyl-, alkylacyl- and diacyl subclassesin EPG in intestinal mucosa of animals fed control or treatment diets.

FIG. 3 shows the fatty acid composition of alkenylacyl, alkylacyl anddiacyl subclasses in CPG in intestinal mucosa of animals fed controldiet or treatment diets.

FIG. 4 shows the fatty acid composition of alkenylacyl, alkylacyl anddiacyl subclasses in EPG in intestinal mucosa of animals fed controldiet or treatment diets.

FIG. 5 shows total content of GG (A), SM (B) and cholesterol (C) inintestinal microdomains after feeding different diets for 2 wks.

FIG. 6 illustrates total content of GD3 (A) and PAF (B) in intestinalmicrodomains after feeding different diets for 2 wks.

FIG. 7 shows the ratio of cholesterol/GG (A) and cholesterol/SM (B) inintestinal microdomains after feeding different diets for 2 wks.

FIG. 8 shows caveolin content determined by western blotting (A), andthe intensity of blots (B) in intestinal microdomains fed control dietor treatment diets for 2 wks.

FIG. 9 illustrates the total content of a) gangliosides and b)phospholipids in the retina of control and treatment groups.

FIG. 10 illustrates immunofluorescent detection of GM3 localization.

FIG. 11 illustrates immunofluorescent detection of GD3 localization.

FIG. 12 shows the effect of dietary treatment on total content ofgangliosides in (A) the intestinal mucosa, (B) plasma and (C) brain foranimals fed either the control or experimental diet for two weeks.

FIG. 13 illustrates the effect of dietary treatment on cholesterolcontent in (A) the intestinal mucosa, (B) plasma and (C) brain ofanimals fed either the control or experimental diets for two weeks.

FIG. 14 shows the effects of dietary treatment on the ratio ofcholesterol to ganglioside in (A) the intestinal mucosa, (B) plasma and(C) brain of animals fed either the control or experimental diets fortwo weeks.

FIG. 15 illustrates the composition of GM3 and GD3 in microdomains ofrat intestine.

FIG. 16 illustrates the composition of PAF and DG in microdomains of ratintestine. Both PAT and DG are reduced with a GG diet.

FIG. 17 illustrates the caveolin content of microdomains for animals feda control, PUFA or GG diet.

FIG. 18 illustrates that feeding a diet high in ganglioside resulted indecreased plasma cholesterol and triglyceride.

DETAILED DESCRIPTION

Generally, the present invention provides a formulation for mediatinginflammation, for example, inflammation of the intestine, of the retina,or of neural tissue other than the retina. Further, a formulation isprovided for lowering blood cholesterol.

The invention is based in the discovery of a ganglioside containingcomposition, such as a milk derived dietary component, that altersinflammation mediators and has an effect in cholesterol lowering.Inflammation mediation occurs particularly in the intestine, the retinaor other neural tissue. The invention is also particularly useful intreating or preventing inflammatory diseases and in reducing bloodcholesterol levels, possibly through decreased intestinal absorption ofcholesterol.

According to embodiments of the invention, a formulation is providedthat is effective in mediating inflammation. Inflammatory states to bemediated include inflammation of the intestine, retina or neuronaltissue, and by “mediating” it is meant preventing or treating aninflammatory disease. For example, such diseases may includeinflammatory bowel disorders, disorders arising from allergic responses,and diseases involving epithelial surface responses. Gastroenteritis,enteric infections, enterocolitis, and necrotizing enterocolitis are allexamples of such inflammatory conditions that may be alleviatedaccording to the invention. Infants may be particularly susceptible tosuch conditions.

According to an embodiment of the invention, a formulation is providedfor reducing plasma cholesterol level.

The formulations according to the invention may include a gangliosidesuch as GD3, GM1, GM2, GM3, GD1b, NANA, and sialic acid. The formulationmay be in the form of a supplemented liquid or food, such as infantformula or infant foods. An exemplary formulation may a totalganglioside composition made up of the following individual components:about 80% GD3, 9% GD1b, and 5% GM3 on a weight/weight basis. A furtherexemplary formulation may have one predominant ganglioside, for example:GD3, GM1, GM2, GM3, GD1b, NANA, and sialic acid, that comprises morethan 50% of the total ganglioside content.

An embodiment of the invention also relates to a method for mediatinginflammation in a subject in need thereof comprising the step ofproviding at least one ganglioside to the subject for oral consumption.The use of at least one ganglioside for preparation of a medicament fororal consumption to mediate inflammation in a subject in need thereof isalso encompassed by an embodiment of the invention.

Further, an embodiment of the invention encompasses a method forreducing blood cholesterol in a subject in need thereof comprising thestep of providing at least one ganglioside to the subject for oralconsumption. Additionally, the invention provides for the use of atleast one ganglioside for preparation of a medicament for oralconsumption to reduce blood cholesterol in a subject in need thereof.

Experiments were done to assess the effects of dietary gangliosides, forexample: components isolated from milk, on a variety of parametersindicative of or causal in mediating an inflammatory oranti-inflammatory response. These experiments lead to the identificationof gangliosides responsible for prophylactic and/or therapeutic effects.

According to the invention, a ganglioside fraction, for example afraction derived from milk, herein referred to as “Fraction A” may beused. Other sources of gangliosides, such as from dairy products orsynthetic sources, may also be used in preparing the formulationsaccording to the invention.

The dosage amount of the ganglioside formulation according to theinvention that may be used for oral delivery can easily be determined byone of skill in the art. A daily or one-time only minimum dosage may befrom microgram to milligram quantities. A higher level may have agreater effect where the exposure and likelihood of infection isincreased. A formulation in food or fluid form having from 1 to 1000 ppmof one or more ganglioside can be delivered to a subject in needthereof. A concentration falling outside of this range may also be used,and no upper limit is required because the formulation does not displaytoxicity, and is not know to be toxic.

To accomplish an inflammation mediating effect or a cholesterol loweringeffect, a typical dosage for adults may be from about 100 mg to about 1g of ganglioside per person per day, based on an adult body weight ofabout 70 kg. However, it is possible to deliver gangliosides in aquantity outside of this range, for example from about 10 mg to about 10g may be effectively delivered to an individual in need thereof.

For an infant having a typical body weight of about 3.5 kg, a level ofgangliosides that may be delivered in order to accomplish aninflammation mediating effect may range from about 10 to about 50 mg perday per infant. However, it is possible to deliver gangliosides in aquantity outside of this range, for example from about 1 mg to about 100mg may be delivered in order to accomplish an inflammation mediatingeffect.

Fraction A may be prepared by crude processes, or may be obtainedcommercially from a source such as New Zealand Dairy, New Zealand.Fraction A is of variable lipid composition, for example, approximately80% GD3; 9% GD1b, and 5% GM3 by weight, the remaining 6% being comprisedof other gangliosides. Fraction B, higher in GM3 can also be prepareddepending on the milk used for ganglioside isolation.

Other molecular forms of this complex lipid may be derived, according tothe invention, with similar or even greater bioactive characteristics.According to one possible composition of the formulation used in theinstant invention, the fraction may contain one or more gangliosides,such as for example GD3, GM1, GM2, GM3, GD1b, NANA, and sialic acid andis bioactive against Giardia producing very high kill rates.

Certain components of the formulation of the invention can be isolatedfrom components of the present food supply, and thus would not need“drug” approval to be added to or to enriched new foods.

Ganglioside supplementation, or supplementation of a lipid fractioncontaining ganglioside can be used to supplement or fortify existingfoods, such as in infant formulas, baby foods, baby cereals, andfollow-on formulas which may be used for children up to about 18 monthsof age. Further, supplementation may also be useful in juices or otherfluids packaged particularly for toddlers or older children, or incereals as a coating or powdered sprinkle. Such foods may advantageouslybe those which are appealing to children, as this could be used to treator prevent inflammatory disease at an early age, or to mediateinflammation.

Foods appealing to adults may also be supplemented or fortified withgangliosides or ganglioside-containing fractions in order to targetindividuals requiring mediation of an inflammatory response. The use ofsuch foods, in treating or preventing inflammatory diseases or to lowerblood cholesterol level, is also encompassed by the present invention.

In addition to being supplemented into food, the formulation may beprovided in a liquid, gel, powder, tablet, pill or capsule form. Tablet,pill or capsule form may appeal to older children and adults, and wouldavoid the need to consume a food or beverage.

The supplement may also be added to pet foods or supplements, or tofoods directed to other domesticated animals. In some instances, it mayalso be desirable to supplement the formulation to livestock. Such a useof gangliosides would be helpful in mediating an inflammatory responsein animals in need thereof.

EXAMPLE 1 Milk Fraction A Containing Gangliosides

Table 1 provides the composition of Fraction A, illustrating the amountof total lipid, calcium and lactose present in 100 g of Fraction A on adry weight basis. The ganglioside and phospholipid content of the lipidfraction is broken down into specific components. In Table 1, allabbreviations used are those defined previously, and additionally: % GGmeans percent of total gangliosides; % PL means percent of totalphospholipids; x-1, x-2 and x-3 are gangliosides; LPC:lysophosphatidylcholine; SM:sphingomyelin; PC: phosphatidylcholine; LPE:lysophosphatidylethanolamine; PS:phosphatidylserine; PI:phosphatidylinositol; PE: phosphatidylethanoloamine.

TABLE 1 Composition of Fraction A Fraction A 100 g Total Lipids(g) 23.00(g) (% GG) Gangliosides (as NANA amt) 0.82 GM3 4.50 x-1 4.60 x-2 0.80GD3 79.90 GD1b 9.00 x-3 1.20 (g) (% PL) PL (as ‘P’) 0.49 LPC 0.036 7.3SM 0.013 2.7 PC 0.012 2.5 LPE 0.093 19.0 PS 0.149 30.4 PI 0.136 27.8 PE0.050 10.2 0.49 99.9 Neutral lipid  0.04 Cholesterol  0.08 Ca (g) 10.00Lactose (g) 65-70

EXAMPLE 2 Separation of Gangliosides from Fraction A

Fraction A, having the composition described above in Table 1, Example1, was obtained and gangliosides were separated therefrom using thefollowing method. The separated ganglioside fraction so obtained may beused in a supplementation regime according to the invention.Alternatively, individual gangliosides obtained from the separatedfraction may be used in a supplementation regime according to theinvention.

Total lipids were extracted from the ganglioside enriched preparation ofFraction A using the Folch method (Folch and Sloane-Stanley, 1957). Theganglioside-containing upper phase was transferred and the lower phasewas washed once with Folch upper phase solution(chloroform/methanol/water, 3/48/47 by vol.). The combinedganglioside-containing fractions were passed through Sep-Pak™ C18reverse-phase cartridges (Waters Corporation, Milford, Mass., USA),eluted with methanol and chloroform and methanol 2:1 (v/v), and driedcompletely under vacuum at 23° C. using a rotary evaporator. Ganglioside(NANA) content was measured as described by Suzuki (1964).

EXAMPLE 3 Dietary Gangliosides Increase Content of Ether PhospholipidsContaining 20:4n-6 and 22:6n-3 in the Rat Intestine

In this Example, the effect of dietary gangliosides on the content ofether phospholipids (EPL) in intestinal mucosa was observed. MaleSprague-Dawley rats (18-day old) were fed a semi-purified dietconsisting of 20% fat as a control diet. Two experimental diets wereformulated by adding either 0.1% (w/w) gangliosides (GG diet) or 0.2%(w/w) sphingomyelin (SM diet) to the control diet. Fatty acid methylesters from the alkenylacyl, alkylacyl, and diacyl subclasses ofphospholipids were measured to determine total and relative percentageof EPL comprising the choline phosphoglyceride (CPG) and ethanolaminephosphoglyceride (EPG) fractions. The GG diet was shown to increase theoverall levels of EPL in both CPG and EPG in intestinal mucosa as wellas increasing the PUFA content of the EPL class, specifically in 20:4n-6and 22:6n-3. As a result of the increase in PUFA content, the ratio ofSFA to PUFA in both CPG and EPG was reduced in animals fed the GG diet.The effects of the SM diet were similar but of lesser magnitude than theGG diet. Enhanced EPL content may suppress carcinogenesis, inflammationand lipid oxidative processes in the developing intestine.

Materials and Methods

Animals and diets. The protocol for this study was approved by theAnimal Care Committee at the University of Alberta, Canada. MaleSprague-Dawley rats (18-day old, 40±4.5 g) were housed in polypropylenecages and maintained at a constant room temperature of 23° C. and a 12 hlight/dark cycle for 2 weeks. Animals had free access to water and wererandomized to be fed one of three semi-purified diets containing 20%(w/w) fat (Table 2). The composition of basal diet is reported elsewhere(Clandinin et al., 1980). The fat in the control diet was a blend oftriglycerides reflecting the overall fat composition of an infantformula. Two experimental diets were formulated by adding eithersphingomyelin (0.2% w/w, Sigma, Mo., USA; SM diet) or a gangliosideenriched lipid powder (0.1% w/w, New Zealand Dairy, New Zealand; GGdiet) to the control diet. The ganglioside enriched lipid powderconsisted of about 45-50% (w/w of total lipid) as phospholipid and15-20% (w/w of total lipid) as gangliosides. The ganglioside fractioncontained about 80% (w/w) GD3 and GD1b, GM3 and other gangliosides (9, 5and 6% w/w, respectively). The fatty acid composition of experimentaldiets was quantitatively analyzed by gas liquid chromatography (GLC,Varian Vista 3400CX). The fatty acid composition was consistent amongthe three diets: 18:1n-9 (50%), 18:2n-6 (20%), 16:0 (16%), 18:0 (8%),18:3n-3 (2.8%) and other fatty acids (3.2%). The control diet andexperimental diets provided an n-6 to n-3 ratio of 7.1. The cholesterolamount was low (<0.35%, w/w of total lipid). Overall the GG dietcontained 19.6% triglycerides, 0.1% gangliosides, 0.2% phospholipid and0.07% cholesterol (Table 2). Body weight and food intake was measuredevery other day throughout the experiment.

TABLE 2 Composition of experimental diets¹ Control SM GG Basal diet(g/100 g) 80.0 80.0 80.0 Triglyceride 20.0 19.8 19.6 Sphingomyelin — 0.2 tr² Ganglioside — — 0.1 Phospholipid — — 0.2 Cholesterol — — tr¹The composition of the control diet are referred from Clandinin et al.,1980, representing an existing infant formula. ²tr presents traceamount.

Collection of intestinal mucosa. After anesthetizing animals withhalothane, the small intestine (jejunum to ileum) was excised. Theintestine was washed with 0.9% cold saline solution to remove visiblemucus and debris, opened, and moisture was carefully removed. Intestinalmucosa was scraped off with a glass slide on an ice cold glass plate.All samples were weighed and kept in a −70° C. freezer until used.

Lipid extraction and phospholipid separation. Total lipid was extractedby using the Folch method (Folch et al., 1957). For extracting totallipid, the intestinal mucosa was washed twice with Folch lower phasesolution (chloroform/methanol/water, 86:14:1, v/v/v). The lower phaselipid was pooled, dried and then dissolved in chloroform:methanol (2:1,v/v). Extracted lipid was applied onto pre-coated silica gel “H” thinlayer chromatography (TLC) plates (Analtech, Newark, Del.) and developedin the solvent (chloroform/methanol/2-propanol/0.25% KCl/triethylamine,45:13.5:37.5:9:27, v/v/v/v/v) to separate individual phospholipidclasses. After spraying with 0.1% ANSA (anilino naphthalene sulfonicacid) and identifying CPG and EPG bands under UV light, the two bandswere scraped into test tubes.

Fatty acid composition and quantification. Solid phases containing CPGand EPG were eluted with 5 mL of chloroform:methanol (2:1) and thendried under N₂. The dried phospholipids were dephosphorylated withphospholipase C (B. cereus, Sigma Chemical Co., St. Louis, Mo.) at 37°C. for 2 hr in a solution of 1 mL diethyl ether, 4 mL 17.5 mM Trisbuffer (pH 7.3), and 1.3 mL 1% CaCl₂ (Bernett et al., 1985). Afterextraction of the hydrolyzed lipids with diethyl ether and petroleumether, lipids were acetylated in 0.1 mL of pyridine with 0.5 mL ofacetic anhydride at 80° C. for 1 hr. The acetylated derivatives ofalkenylacyl, alkylacyl and diacyl phospholipids were extracted withchloroform/methanol (2:1) solution, applied onto a silica gel highperformance TLC plate (HPTLC, Whatman Inc, Clifton, N.J.) and developedin petroleum ether: diethyl ether: acetic acid, (90:10:1, v/v/v) to amigration distance of 10 cm from the solvent line, followed by a seconddevelopment in toluene (Holub et al., 1987). The plate was sprayed withANSA and visualized under UV light. Three subclasses of the CPG and EPGfractions scraped from the plate were then methylated in3N-methanolic-HCl (Supelco, Pa., USA) for 16 hr at 70° C. with a knownamount of heptadecanoic acid as an internal standard. The fatty acidcomposition of each of the six fractions was analyzed by GLC equippedwith a flame ionization detector and BP-20 fused capillary column (SGE,Australia). The flow rate of helium gas was 1.6 mL/min and the oven,injector and detector temperatures were 200° C., 250° C., and 250° C.,respectively. Since only acylated fatty acids (not ethers) can beconverted to fatty acid methyl esters for GLC analysis, the fatty acidamount measured in EPL only represents one-half of the total. Thus, thisvalue was multiplied by two to have total and molecular percentage ofEPL.

Statistical analysis. The values were shown as means±standard deviationfrom eight animals, with a few exceptions indicated. Significantdifferences between the control group and experimental groups weredetermined by one-way analysis of variance (ANOVA) with the SAS system.Effects of diet treatment were determined by a Duncan multiple rangetest at significance levels of P<0.05, P<0.01, P<0.001 or P<0.0001.

Results

Animal growth and intestinal mucosa. Initial body weight of animals,weight after two weeks of diet treatment was not significantly differentbetween experimental and control groups. Intestinal mucosal weight andlength was not affected by dietary treatment. Food consumption was alsonot influenced by diet treatment.

Fatty acid content corresponding to alkenylacyl, alkylacyl and diacylphospholipids in CPG and EPG. Animals fed the GG diet presentedsignificantly higher amount of fatty acids from alkenylacyl-CPG,alkenylacyl-EPG and alkylacyl-EPG in comparison with control animals(56%, 77% and 54% increases, respectively; Table 3). The highest changein fatty acid content relative to the control was observed inalkenylacyl-EPG (P<0.0008). In animals fed dietary gangliosides, asignificant decrease in diacyl-CPG occurred, whereas no change wasobserved in diacyl-EPG. Animals fed the SM diet exhibited a similarincrease in fatty acid content corresponding to alkylacyl-GPE, but nochange occurred in the other subclasses of GPC and GPE. In the totalfatty acid content of EPL (alkenylacyl and alkylacyl together), the GGdiet produced significant increase, by 36% and 66% respectively,comprising CPG and EPG phospholipids in intestinal mucosa (Table 3).Feeding animals the SM diet only increased total EPL in EPG by 42%compared to control animals.

TABLE 3 Fatty acid content of alkenylacyl, alkylacyl and diacylsubclasses in CPG and EPG from intestinal mucosa of animals fedexperimental diets¹ Effect of Subclass (μg/g tissue) Control SM GG diet(P<) Total EPL-CPG² 31.8 ± 6.7^(b) 37.8 ± 8.7^(ab) 43.3 ± 8.2^(a) 0.05Alkenylacyl-CPG 14.0 ± 3.6^(b) 17.0 ± 3.0^(ab) 21.8 ± 7.2^(a) 0.05Alkylacyl-CPG 17.8 ± 3.9 20.8 ± 7.5 21.5 ± 4.8 Diacyl-CPG 1.86 × 10³ ±344^(a) 1.61 × 10³ ± 469^(ab) 1.22 × 10³ ± 306^(b) 0.01 Total EPL-EPG68.1 ± 7.9^(c) 97.1 ± 15.3^(b)  113 ± 20^(a) 0.001 Alkenylacyl-EPG 34.4± 7.7^(b) 45.2 ± 14.5^(b) 60.9 ± 12.3^(a) 0.0008 Alkylacyl-EPG 33.7 ±4.4^(b) 51.9 ± 10.6^(a) 51.8 ± 10.7^(a) 0.0006 Diacyl-EPG  774 ± 99  731± 292  801 ± 174 ¹Mean ± SD from 7, 8, and 7 animals, for the Control,SM and GG group, respectively. ²Fatty acid content of total EPL(alkenylacyl and alkylacyl together) in CPG or EPG.

Changes in relative amounts of SFA, MUFA, and PUFA in alkenylacyl,alkylacyl and diacylphospholipids comprising CPG and EPG classes. AsPUFA levels in alkylacyl-CPG increased in animals fed the GG diet, therewas a concomitant decrease in the ratio of SFA to PUFA in alkylacyl-CPG(2.6 versus 6.8, P<0.05; FIG. 1). In contrast, in diacyl-CPG fractions,animals fed the GG diet exhibited an increase in the ratios of SFA toMUFA (5.3 versus 3.6, P<0.0007) and SFA to PUFA (1.5 versus 1.1,P<0.05).

FIG. 1 illustrates the ratio of SFA to MUFA (white columns) and SFA toPUFA (black columns) in alkenylacyl-, alkylacyl- and diacyl subclassesin CPG in intestinal mucosa of animals fed control or treatment diets.Values are means±SD of 7, 8 and 7 animals for the control, SM and GGdiet, respectively. Letters represent a significant difference betweengroups at P<0.05, except for the ratio of SFA to MUFA in the diacylsubclass at P<0.0007.

Changes in relative amounts of SFA, MUFA, and PUFA in the threesubclasses of EPG are illustrated (FIG. 2). In the GG diet group,alkenylacyl-EPG and alkylacyl-EPG exhibited significantly lower ratiosof SFA to PUFA compared to the controls (0.6 versus 1.3 foralkenylacyl-GPE, P<0.005, and 0.5 versus 1.2 for alkylacyl-GPE, P<0.05).There was also a decrease in the SFA/MUFA ratio (1.8 versus 2.5) in thealkylacyl group. Animals fed the SM diet exhibited a lower SFA/PUFA inthe both EPL subclasses compared to animals fed the control diet, buteffects were more subtle than that of the GG diet. There was no observedeffect of SM or GG treatments on the diacyl-EPG class.

FIG. 2 shows the Ratio of SFA to MUFA (white columns) and SFA to PUFA(black columns) in alkenylacyl-, alkylacyl- and diacyl subclasses in EPGin intestinal mucosa of animals fed control or treatment diets. Valuesare means±SD of 7, 8 and 7 animals for the control, SM and GG diet,respectively. Letters represent a significant difference between groupsat P<0.005, except for the ratio of SFA to PUFA in the alkylacylsubclass at P<0.02.

Changes in relative composition of alkenylacyl, alkylacyl anddiacylphospholipids comprising CPG and EPG. Animals fed the GG dietexhibited higher levels of alkenylacyl-CPG (3.3% versus 1.6%) andalkylacyl-CPG (3.2% versus 2.1%) with correspondingly lower levels ofdiacyl-CPG (93.5% versus 96.3%), compared to animals fed the controldiet (Table 4). No effect of the SM diet was observed on the relativecomposition of CPG in the mucosa. Feeding animals the GG diet achievedhigher levels of alkenylacyl-EPG (12.5% versus 7.4%) and alkylacyl-EPG(10.9% versus 7.3%) with correspondingly lower levels of diacyl-EPG(76.6% versus 85.3%), compared to animals fed the control diet. Animalsfed the SM diet showed a relative increase in alkenylacyl-EPG, but theeffect was less pronounced than in those fed the GG diet. There was noeffect of the SM diet on alkylacyl-EPG. Relative to total phospholipids,animals fed the GG diet also exhibited a marked increase in total EPL(alkenylacyl and alkylacyl together) from 3.7% to 6.5% in CPG and from14.7% to 23.4% in EPG (Table 4). Feeding the SM diet resulted in arelative increase in EPG, but not in CPG.

TABLE 4 Percentage of alkenylacyl, alkylacyl and diacyl subclasses inCPG and EPG from intestinal mucosa of animals fed experimental diets¹Subclass Effect of (%, w/w) Control SM GG diet (P<) Total-EPL-CPG²  3.7± 0.8^(b)  4.8 ± 1.1^(b)  6.5 ± 1.8^(a) 0.006 Alkenylacyl-CPG  1.6 ±0.4^(b)  2.2 ± 0.8^(ab)  3.3 ± 1.3^(a) 0.05 Alkylacyl-CPG  2.1 ± 0.5^(b) 2.6 ± 0.6^(ab)  3.2 ± 0.8^(a) 0.05 Diacyl-CPG 96.3 ± 0.8^(a) 95.2 ±1.1^(a) 93.5 ± 1.8^(b) 0.007 Total-EPL-EPG 14.7 ± 1.6^(b) 20.4 ± 5.4^(a)23.4 ± 3.2^(a) 0.001 Alkenylacyl-EPG  7.4 ± 1.5^(c) 10.2 ± 2.1^(b) 12.5± 1.4^(a) 0.0001 Alkylacyl-EPG  7.3 ± 1.1^(b) 10.2 ± 3.7^(ab) 10.9 ±2.5^(a) 0.05 Diacyl-EPG 85.3 ± 1.6^(a) 79.6 ± 5.4^(b) 76.6 ± 3.2^(b)0.002 ¹Mean ± SD from 7, 8, and 7 animals, for the Control, SM and GGgroup, respectively. ²Percent of total EPL (alkenylacyl and alkylacyltogether) relative to total phospholipids in CPG or EPG. Changes infatty acid composition of alkenylacyl-, alkylacyl- and diacyl-CPG

Changes in fatty acid composition of alkenylacyl-, alkylacyl- anddiacyl-CPG. In comparison with control animals, animals fed the GG dietdid not show significant change in the fatty acid composition ofalkenylacyl-CPG except for 14:1, which decreased (FIG. 3). Animals fedthe GG diet showed an increase in 20:4n-6 as compared to animals fed theSM diet. Decrease occurred in alkylacyl-CPG content of 18:0, 24:0 and24:1 for animals fed the GG diet as well as a distinct increase in22:6n-3 (P<0.0007). The SM diet produced a decrease in 18:0 and anincrease in 22:6n-3 content. In diacyl-CPG, higher levels of 16:0 and18:0 and lower levels of 18:1n-9 and 18:2n-6 were observed in animalsfed the GG diet compared to control animals. Similar trends in 16:0,18:1n-9 and 18:2n-6 were observed for animals fed the SM diet.

Changes in fatty acid composition of alkenylacyl-, alkylacyl- anddiacyl-EPG. The fatty acid composition of alkenylacyl-, alkylacyl, anddiacyl-EPG in intestinal mucosa of animals fed experimental diets isillustrated (FIG. 4). Animals fed dietary gangliosides showed higherlevels in alkenylacyl-EPG of 22:4n-6 (100% increase, P<0.001) and22:6n-3 (71% increase, P<0.001) compared to controls, and lower levelsof 16:0 and 18:0. Animals receiving the SM diet exhibited a similarchange in 22:4n-6, 22:6n-3 and 16:0 fatty acid content, but the effectwas smaller than observed for animals fed the GG diet. In alkylacyl-EPG,higher content of 20:4n-6, 22:4n-6, and 22:6n-3 (36%, 87% and 77%increases, respectively) was observed in animals fed the GG diet with aconsiderable reduction of saturated fatty acids 16:0, 18:0 and 24:0relative to animals fed the control diet. In diacyl-EPG, dietarygangliosides increased the content of 20:4n-6 by 36%, relative tocontrols.

Changes in total SFA, MUFA and PUFA content of alkenylacyl-, alkylacyl-and diacyl-CPG. The fatty acid content of SFA, MUFA and PUFA inalkenylacyl-, alkylacyl-, and diacyl-CPG is shown (FIG. 3). Feedinganimals the GG diet increased total PUFA content in alkylacyl-GPC, whichrose by 63% compared to animals fed the control diet. No changesoccurred in MUFA and SFA for this lipid subclass. Animals fed the GGdiet had lower content of PUFA and MUFA and increased content of SFA indiacyl-CPG than observed for control animals. For animals given the SMdiet, higher level of PUFA in alkylacyl-CPG lipids was found. Theseanimals also exhibited lower MUFA and higher SFA contents in diacyl-CPGcompared to controls.

Changes in total SFA, MUFA and PUFA content of alkenylacyl-, alkylacyl-and diacyl-EPG. Animals fed the GG diet exhibited increased content ofPUFA in the three EPG lipid subclasses compared to control animals,(increase of 41% in alkenylacyl, 41% in alkylacyl and 30% in diacyl-EPG;FIG. 4). The increase in PUFA content was accompanied with a decrease inSFA content in alkenylacyl and alkylacyl-EPG. No change was observed inMUFA in alkenylacyl, alkylacyl or diacyl phospholipids. Feeding of theSM diet also resulted in increased content of PUFA and decreased contentof SFA in alkenylacyl-EPG and alkylacyl-EPG compared to controls. Noeffect of the SM diet was detected in diacyl-EPG.

Changes in relative amounts of SFA, MUFA, and PUFA in alkenylacyl,alkylacyl and diacylphospholipids comprising CPG and EPG classes. AsPUFA levels in alkylacyl-CPG increased in animals fed the GG diet, therewas a concomitant decrease in the ratio of SFA to PUFA in alkylacyl-CPG(2.6 versus 6.8, P<0.05; FIG. 1). In contrast, in diacyl-CPG fractions,animals fed the GG diet exhibited an increase in the ratios of SFA toMUFA (5.3 versus 3.6, P<0.0007) and SFA to PUFA (1.5 versus 1.1,P<0.05).

Changes in relative amounts of SFA, MUFA, and PUFA in the threesubclasses of EPG are illustrated (FIG. 2). In the GG diet group,alkenylacyl-EPG and alkylacyl-EPG exhibited significantly lower ratiosof SFA to PUFA compared to the controls (0.6 versus 1.3 foralkenylacyl-GPE, P<0.005, and 0.5 versus 1.2 for alkylacyl-GPE, P<0.05).There was also a decrease in the SFA/MUFA ratio (1.8 versus 2.5) in thealkylacyl group. Animals fed the SM diet exhibited a lower SFA/PUFAratio in the both EPL subclasses compared to animals fed the controldiet, but effects were more subtle than that of the GG diet. There wasno effect of SM or GG treatments observed on the diacyl-EPG class.

Discussion

Previous studies (Merrill et al., 1997; Schmelz et al., 1994; Ogura etal., 1988) showing a possible mechanism to convert gangliosides to EPLin vivo do not explain whether dietary gangliosides can be absorbed andthen used for EPL synthesis. The present study confirms that dietarygangliosides can be utilized for biosynthesis of EPL in developing ratintestinal mucosa. There are two possible mechanisms which may explainthis effect. Firstly, it is assumed that hexadecanal, a derivative ofgangliosides, is directly utilized for the synthesis of EPL as aprecursor of ether-linked fatty alcohols [Merrill et al., 1997; Schmelzet al., 1994]. This hypothesis is supported by a study showing thatintraperitoneal injection of [³H]GM1, labeled at the C-3 position ofsphingosine, is converted into alkylacyl-EPG in the mouse liver [Oguraet al., 1988]. Secondly, a possible indirect mechanism is that reductionof cholesterol in intestinal cells caused by dietary gangliosides mayincrease EPL synthesis or uptake. Earlier studies show that a decreasein cholesterol content increase EPL uptake in human leukemia cell lines[Diomede et al., 1990; Diomede et al., 1991]. Since experiments in ourlaboratory have shown a significant decrease in total cholesterolcontent in the intestine of animals fed the GG diet, it is logical toassume that dietary gangliosides may increase EPL synthesis bydecreasing intestinal cholesterol content.

Several studies have shown that EPL is enriched in neuronal tissues andmay play a functional role in neuronal development [Horrocks et al.,1972; Hollan et al., 1998; Sindelar et al., 1999; and Farooqui et al.,2000]. For example, EPL, especially plasmalogen, dramatically increasedduring 3-90 days in rat cerebellum development [Leray et al., 1990] andwithin one year of birth in the human brain [Rouser et al., 1968].Plasmalogen levels are also significantly higher during celldifferentiation in NILE-115 neuroblastoma cells [Murphy et al., 1993].EPL is a major phospholipid present during synaptogenesis andmyelination [Leray et al.,1990; Rouser et al., 1968] in which EPL mayact as an endogenous antioxidant for membrane peroxidation [Sindelar etal., 1999; Farooqui et al., 2000; and Reiss et al., 1997]. The presentresults suggest that dietary gangliosides may enhance development of theenteric nerve system during neonatal gut development by providing ameans, for increasing EPL content.

Another possible implication of elevated EPL level is an effect onanti-inflammatory response. Alkylacylglycerol, an analogue ofdiacylglycerol (DG) which is derived from EPL by phospholipase C (PLC),is known to have a potent inhibitory effect on lipoxygenase [Bauldry etal., 1988] and cytosolic phospholipase A2 (cPLA2) activity [Nixon etal., 1997], both of which stimulate an inflammatory response.Alkylacylglycerol also decreases leukotriene B4 and5-hydroxyeicosatetraenoic acid (5-HETE) production [Bauldry et al.,1991] by inhibiting PKC activity [Parker et al., 1987] compared to DG.Gangliosides also inhibit PLA2 activity [Basavarajappa et al., 1997].This hypothesis is supported by our recent study demonstrating thatdietary gangliosides decrease platelet activating factor (PAF) and DG inintestinal microdomains. Alkylacyl-phospholipids are mostly localized atthe inner membrane [Record et al., 1984], Our results showing a 52% and49% increase in the level of alkylacyl-CPG and -EPG, respectively,suggest that dietary gangliosides may enhance the inner localization ofEPL. This localization of the alkylacyl subclass of EPL at the innermembrane may influence anti-inflammatory response [Bauldry et al., 1988;Nixon et al., 1997; Bauldry et al., 1991; and Parker et al., 1987] bydown-regulating cytosolic enzymes and proteins, such as cPLA2 and PKC,known to be inflammatory mediators.

Higher levels of PUFA in EPL in animals fed the GG diet may haveresulted from a decrease in total cholesterol content in intestinalmucosa which is known to cause a corresponding increase of α-5 and α-6desaturase activities [Leikin et al., 1988]. Finding that dietarygangliosides promoted a higher level of 20:4n-6, 22:4n-6 and 22:6n-3 inEPG raises the question of whether these PUFA may serve a particularfunction in the enteric nervous system as in other neuronal tissues. Forexample, 22:6n-3 protects retinal photoreceptors by delaying the onsetof apoptosis and activates photoreceptor differentiation, promotingopsin expression and inducing apical differentiation in these neurons[Polit et al., 2001]. Supplementation of LC-PUFAs resulted in aresistance against NMDA-induced excitotoxic degeneration of cholinergicneurons in infant rats [Hogyes et al., 2003].

The present study demonstrates that dietary gangliosides increase totaland relative percentage of EPL and the PUFA content of EPL in intestinalmucosa during neonatal development. These results suggest that dietarygangliosides influence gut development and protection by enhancing EPLcontent which may have a preventative role in carcinogenesis,inflammation, and lipid oxidation. Further investigation is needed todetermine if dietary ganglioside also affects the synthesis of EPL inneuronal tissues such as brain, retina and the myenteric plexus in theintestine.

EXAMPLE 4 Diet-Induced a Decrease in the Ratio of Cholesterol toSphingolipids Attenuates the Caveolin and Inflammatory Mediator Contentin Microdomains of the Rat Intestine

Membrane microdomains rich in cholesterol and sphingolipids includinggangliosides are known as cellular binding sites for various pathogens.Cholesterol depletion inhibits the cellular entry of pathogens and alsoreduces inflammatory signals by disrupting microdomain structure. Ourprevious study showed that dietary gangliosides increased gangliosideswhile decreasing cholesterol in the intestinal mucosa. We hypothesizedthat diet-induced reduction in the cholesterol content in intestinalmicrodomains disrupts microdomain structure resulting in reducedpro-inflammatory signals. To test this hypothesis, Sprague-Dawley rats(1 8-day old) were fed semi-purified diets for 2 wks. The control dietcontained 20% triglyceride. Experimental diets were formulated by addingeither 0.1% ganglioside enriched lipid (GG diet, w/w of diet) or 1.5%,w/w total fatty acids, polyunsaturated fatty acid (PUFA diet, 1% 20:4n-6and 0.5% 22:6n-3) as triglyceride to the control diet. The gangliosideenriched lipid contained 70-80% GD3 among the total gangliosidefraction. Levels of cholesterol, ganglioside, caveolin expression andpro-inflammatory mediators, platelet activating factor (PAF) anddiglyceride (DG) was measured in microdomains. Feeding animals the GGdiet increased GG and decreased cholesterol content in intestinalmicrodomains by 50% and 23%, respectively. These changes resulted in asignificant decrease in the ratio of cholesterol to GG. Increased GD3and decreased GM3 was found in the intestinal microdomains of animalsfed the GG diet in comparison to animals fed the control diet. Caveolincontent was significantly reduced in animals fed the GG diet along withreduction in PAF and DG content in the microdomain. Animals fed the PUFAdiet also showed decreased cholesterol, caveolin, PAF and DG content inintestinal microdomains compared to animals fed the control diet,without change occurring in the sphingolipid profile. It is concludedthat dietary GG decrease the cholesterol/GG ratio, caveolin, PAF and DGcontent in microdomains and may have a potential anti-inflammatoryeffect during gut development.

The objective of this example was to determine if cholesterol reductionin intestinal mucosa by dietary gangliosides consequently inducesdecreases in the ratio of cholesterol/sphingolipids, structuraldisruption of microdomains and ultimately attenuates the level ofpro-inflammatory mediators in developing gut.

Materials and Methods

Animals and Diets. The experiments presented in this example wereapproved by the University of Alberta Animal Ethics Committee. MaleSprague- Dawley rats (18-day-old, n=24), average body weight 41.6±1.6 g,were randomly separated into 3 groups of 8 with 2 or 3 rats housed ineach polypropylene cage. Animals were maintained at a constanttemperature of 23° C. and a 12 h light/dark cycle. Animals had freeaccess to water and one of three semi-purified diets containing 20%(w/w) fat for 2 weeks. The composition of the basal diets fed has beenpreviously reported (Table 5) (Clandinin et al., 1980). Animal bodyweight and food intake were recorded every other day throughout theexperiment. The control diet (CONT diet) fat was a blend oftriglyceride, which reflected the fat composition of an existing infantformula. Fatty acids of the control diet (Jumpsen et al., 1997) werecomposed of about 31% saturated fatty acids, 48% monosaturated fattyacids and 21% polyunsaturated fatty acids providing a ratio of 18:2n-6to 18:3n-3 of 7.1. Two experimental diets were formulated by addingeither polyunsaturated fatty acids such as 1% arachidonic acid (20:4n-6)and 0.5% docosahexaenoic acid (22:6n-3) (PUFA diet, 1.5% w/w, MartekBiosciences, USA) or a ganglioside-enriched lipid (GG diet, 0.1% w/w,New Zealand Dairy, New Zealand) to the control diet. Gangliosideenriched lipid consisted of about 45-50% (w/w) phospholipids and 15-20%(w/w) gangliosides. The cholesterol content was negligible (<0.35% w/wtotal lipid). The ganglioside fraction contained about 80% w/w GD3 andGD1b, GM3 and other gangliosides (GM2, GM1 and GT1b) was 9, 5 and 6%w/w, respectively.

TABLE 5 Composition of Experimental Diets¹ Diet Treatment Control PUFAGG Basal diet (g/100 g) 80.0 80.0 80.0 Triglyceride 20.0 20.0 19.620:4n-6 — 1.0 — 22:6n-3 — 0.5 — Ganglioside — — 0.1 Phospholipid — — 0.2Cholesterol — — tr² ¹The composition of the basal diet has beenpreviously published (Clandinin et al., 1980). The fatty acidcomposition of the control fat blend is similar to that of an infantformula fat mixture (Jumpsen et al., 1997). ²tr presents trace amount.

Collection of Samples. After anesthetizing animals with halothane, thesmall intestine (jejunum to ileum) was excised. The intestine was washedwith ice cold 0.9% saline solution to remove visible mucus and dietarydebris, opened and moisture was carefully removed with a paper towel tomeasure mucosa weight. Intestinal mucosa was scraped off with a glassslide on an ice cold glass plate. All mucosa samples were kept in a −70°C. freezer until extraction.

Sucrose gradient separation of microdomains. Intestinal microdomainswere prepared by ultra-centrifugation of a discontinuous sucrosegradient (Igarashi et al., 2000). Intestinal mucosa was suspended withTME (10 mM Tris-HCl, 1 mM MgCl₂, 1 mM EGTA) solution containing 1 mMphenylmethyl sulfonyl fluoride, 0.001% w/v apotinin and 2% v/v TritonX-100 for 30 min in ice and homogenized with 15 strokes of a Douncehomogeniser with a tight-fitting pestle (Wheaton Scientific, USA). Thehomogenate was adjusted to 45% w/v sucrose by adding the equal vol of90% w/v sucrose and then homogenized again with 5 strokes of the Douncehomogeniser. A 5-35% discontinuous sucrose gradient was overlaid on thehomogenate in 45% w/v sucrose, which left a 45-35-5% sucrose gradientfrom the bottom. After 16 h centrifugation at 70,000×g at 4° C. in aBeckman SW 28 Ti rotor, interface fraction between 5 and 35% sucrose wascollected as the microdomain fraction. Microdomains were washed with TMEsolution and centrifuged twice at 100,000×g for 1 h at 4° C. to removesucrose and Triton X-100. The pellet was resuspended in phosphate buffersolution and used for protein and lipid analysis. Enrichment ofmicrodomains was demonstrated by testing the amounts of cholesterol andgangliosides in the intestinal microdomain compared to the proteinpellet which was soluble in detergent solution. In the intestinalmicrodomains, cholesterol content was 10 fold higher compared to thedetergent soluble proteins. Intestinal gangliosides were exclusivelyfound in the microdomain compared to negative intensity in the detergentsoluble protein pellet by a densitometry assay on TLC plates.

Western Blotting for Caveolin Content. Protein content from microdomainswas measured by QuantiPro BCA™ Assay Kit (Sigma-Aldrich Co. Mo. USA).Approximately 25 mg proteins were dissolved with SDS reducing samplebuffer and loaded onto 15% SDS-PAGE minigels. After transferringproteins onto nitrocellulose membrane (Amersham Pharmarcia Biotech, UK),membranes were blocked with 5% non-fat dried milk in TBS-T (20 mM Tris;pH 7.6; 137mM NaCl; 0.1% Tween-20) for 1 h at room temperature. Theprimary antibody (BD Biosceince, ON. CA), which specifically recognizescaveolin was diluted in TBS-T with 1% non-fat dried milk (1:1000) andincubated for 90 min at room temperature. The membrane was washed threetimes for 10 min each time in TBS-T. The secondary antibody (goatanti-mouse IgG-HRP conjugate; Bio-Rad, Calif. USA) was diluted in 1%non-fat dried milk in TBS-T (1:2000) and incubated for 1 h at roomtemperature. After washing the membrane with TBS-T three times for 10min each time, the caveolin protein was developed by enhancedchemiluminescence (ECL) detection reagent according to the protocolsupplied by Amersham Pharmarcia Biotech, UK. Blot intensity of caveolinwas measured from five animals from each diet group by using an ImagingDensitometer (Bio-Rad, Calif. USA).

Ganglioside Extraction and Purification. Total lipid of the microdomainfraction was extracted using the Folch method (Folch et al., 1957). Forextraction of gangliosides (Svennerholm, 1964), the upper phase wascollected into a test tube and the lower organic phase was washed twicewith the Folch upper phase solution (chloroform/methanol/water, 3/48/47by vol.) to increase GG content isolated from the microdomain fraction.The upper phase gangliosides were pooled and purified by passage throughSep-Pak™ C-18 cartridges (Waters Corporation, Milford, Mass., USA)prewashed with 10 ml of methanol, 20 ml of chloroform/methanol (2/1,v/v), and 10 ml of methanol as described by Williams and McCluer(Williams et al., 1980). The upper phase extract was loaded onto Sep-PakC-18 cartridges. Cartridges were then washed with 20 ml of distilledwater to remove salts and water-soluble contaminants. Gangliosides wereeluted with 5 ml of methanol and 20 ml of chloroform/methanol (2/1,v/v), dried under N₂ gas and then redissolved with 500 ul ofchloroform/methanol (2/1, v/v). Gangliosides were stored at −70° C.until analysis.

Analysis of Total and Individual Ganglioside Content. Total NANA ofgangliosides was measured as described by Suzuki (Suzuki, 1964). Analiquot of the ganglioside sample purified using Sep-Pak C-18 cartridgeswas dried under N₂ gas and dissolved with each of 0.5 ml of distilledH₂O and resorcinol-HCl (Svennerholm, 1957) in screw-capped Teflon-linedtubes. The purple blue color developed by heating was extracted intobutylacetate/butanol (85/15, v/v) solvent. Optical density was read by aspectrophotometer (Hewlett Packard, 8452A) at 580 nm. Forquantification, N-acetyl neuraminic acid (NANA; Sigma, Mo., USA) wasused as a standard and total ganglioside content is presented as NANA.

Individual gangliosides were separated by Silica gel high performancethin layer chromatography (HPTLC; Whatman Inc, Clifton, N.J., USA) usingganglioside standards, GM3, GM2, GD3 and bovine brain gangliosidemixture (Alexis, San Diego, Calif., USA) in a solvent system ofchloroform/methanol/0.2% (w/v) CaCl₂.2H₂O (55/45/10, by vol.).Individual gangliosides were recovered and measured as described above.

Cholesterol Assay. Cholesterol analysis was completed with a test kit(Sigma, Mo., USA).

Analysis of Sphingomyelin (SM) and Platelet Activating Factor (PAF).Total lipid extracted from microdomains was applied onto a silica gel‘H’ TLC plate using chloroform/methanol/2-propanol/0.2%KOH/triethylamine, 45:13.5:37.5:9:27, by vol) and a silica gel ‘G’ TLCplate (Fisher Scientific, Calif.) using chloroform/methanol/water,(65:35:6, by vol) for SM and PAF, respectively. Commercial standards ofSM, PAF, and lyso-PC (Sigma, Mo., USA) were also spotted on the platefor identification. After development, TLC plates were dried, sprayedwith 0.1% ANSA (anilino naphthalene sulfonic acid) and exposed under UVlight to detect SM and PAF. Lipids identified were recovered and lipidphosphate was measured (Itoh et al., 1986).

Analysis of Diglyceride (DG). To measure DG content, extracted lipid wasapplied onto a silica gel ‘G’ TLC plate using a solvent system(petroleum ether/diethyl ether/acetic acid, 80:20:1, by vol). After TLCdevelopment, 1,2-DG and 1,3-DG were exposed to 0.1% ANSA and identifiedunder UV light with commercial standards. Cholesterol was recovered with1,3-DG together as 1,3-DG comigrates with cholesterol. 1,2-DG and 1,3-DGwere methylated with a known amount of heptadecanoic acid (C17:0) as aninternal standard to measure the total fatty acid amount. To removecholesterol from 1,3-DG after methylation, fatty acid methyl esters(FAME) were applied onto a silica gel ‘G’ plate and developed withToluene. Purified FAME was collected, extracted with hexane and injectedinto the gas liquid chromatograph (GLC, Varian Model 3400 CX, CA) tomeasure total fatty acid content in DG. The GLC was equipped with aflame ionization detector and a 25 m BP-20 fused capillary column (SGE,Australia).

Statistical Analysis. Values shown are means±standard deviation (SD).Significant difference between the control group and experimental groupswas determined by one-way analysis of variance (ANOVA) with SAS.Significant effects of diet treatment were determined by a Duncanmultiple range test at a significance level of p<0.05.

Results

Animal Growth and Tissues. The initial and final body weight of animalsafter 2 weeks feeding of experimental diets was not significantlydifferent among Control, PUFA and GG groups. Intestinal mucosal weightand intestinal length was not affected by dietary treatment. Foodconsumption was not influenced by diet (data not shown).

Ganglioside Content and Composition. The effect of dietary gangliosideon total ganglioside content of intestinal microdomains from animals fedeither the CONT or experimental diets for 2 weeks is shown (FIG. 5-A).Animals fed the GG diet had higher ganglioside content in intestinalmicrodomains (up to a 50% increase; P<0.006) when compared to animalsfed the CONT diet.

FIG. 5 illustrates the total content of GG (A), SM (B) and cholesterol(C) in intestinal microdomains after feeding different diets for 2 wks.Intestinal microdomains were prepared by a discontinuous sucrose density(5-35-45%) ultracentrifugation from mucosa homogenates suspended by TMEsolution containing 2% v/v Triton-X 100. GG extracted from the Folchupper phase was purified by Sep-Pak C-18 cartridges and used for colordensitometry analysis of total NANA content at 580 nm (Suzuki 1964). SMfrom the lower phase was identified on ‘H’ TLC plates inchloroform/methanol/2-propanol/0.2% KOH/triethylamine solvent system(45:13.5:37.5:9:27, by vol). Phosphate content in SM was measured by aknown method (Itoh et al., 1986). Cholesterol content was analyzed byusing a test kit. NANA (P<0.006), phosphate (P<0.004) or cholesterol(P<0.02) content in the microdomain was presented as mg/mg protein. Datawere presented as Mean±SD with n=8 animals in each group.

Animals fed dietary ganglioside significantly decreased GM3 compositionin microdomains compared to animals fed the control diet (83.7% to77.7%, w/w; Table 6) while GD3 increased from 4.4% to 8.3% (w/w;P<0.0008). These compositional changes in GM3 and GD3 were accompaniedby a significant change in GD3 content (P<0.002; FIG. 6-A) but not inGM3 content. GM1, GD1a, GD1b and GT1b content was not changed by eitherdiet fed (Table 6). Animals fed the PUFA diet did not exhibit change ineither GG content or composition in microdomains from developinganimals. The present result confirms that dietary gangliosidesignificantly increased total GG content resulting in compositionalchanges such as decreased GM3 and increased GD3 in neonatal intestinalmucosa.

TABLE 6 Composition of Gangliosides in Rat Intestinal Microdomains FedEither Control or Experimental Diets¹ Diet Treatment Control PUFA GGGanglioside² (%)³  GM3 83.7 ± 2.7^(a)  80.3 ± 4.0^(ab) 77.7 ± 3.3^(b )GM1 2.7 ± 0.7 2.1 ± 1.9 2.7 ± 1.4 GD3  4.4 ± 1.0^(b)  4.5 ± 1.4^(b)  8.3± 2.1^(a4) GD1a 3.5 ± 1.0 3.6 ± 1.0 3.5 ± 1.2 GD1b 3.5 ± 1.1 5.6 ± 2.14.2 ± 1.9 GT1b 2.2 ± 1.9 3.9 ± 2.1 3.5 ± 0.5 ¹Values are means ± SD of 8rats. Within a row, values with different superscript letters aresignificantly different at P < 0.02. ²Nomenclature was referred fromSvennerholm (Svennerholm 1964) ³Expressed as a % of total NANA inganglioside fraction. ⁴Values are significantly different at P < 0.0008.

FIG. 6 shows total content of GD3 (A) and PAF (B) in intestinalmicrodomains after feeding different diets for 2 wks. Intestinalmicrodomains were prepared by a discontinuous sucrose density (5-35-45%)ultracentrifugation from mucosa homogenates suspended by TME solutioncontaining 2% v/v Triton-X 100. GG extracted from the Folch upper phasewas purified by Sep-Pak™ C-18 cartridges. Individual gangliosides wereseparated by HPTLC in a solvent system of chloroform/methanol/0.2% (w/v)CaCl₂.2H₂O (55/45/10, by vol.). GD3 was recovered and measured asdescribed (Suzuki 1964). Total lipid extracted from microdomains wasapplied onto a silica gel ‘G’ TLC plate using chloroform/methanol/water,(65:35:6, by vol) for PAF. Lipid identified was recovered and phosphatein PAF was measured (43). The amount of GD3 (P<0.01) and PAF (P<0.05) inthe microdomain was presented as mg/mg protein. Data were presented asMean±SD with n=8 animals in each group.

Sphingomyelin Content. Results show that animals fed the GG dietmarkedly increased SM content in microdomains by 57% (P<0.004), comparedto animals fed the CONT diet (FIG. 5-B). No change in SM content in themicrodomain was observed in animals fed either the PUFA diet or the CONTdiet. This result demonstrates that dietary ganglioside directlyincreases SM content in intestinal microdomains in developing animals.

Cholesterol Content of Microdomains. Animals fed the GG diet showedsignificantly lower levels of cholesterol in microdomains, compared toanimals fed the CONT diet for 2 wks (FIG. 5-C). Animals fed the PUFAdiet exhibited a lower level of cholesterol in the microdomain, but theeffect on cholesterol reduction in the microdomain was smaller than thatobserved after feeding the GG diet. This result suggests thatcholesterol reduction induced by dietary ganglioside in intestinalmucosa is probably due to a decrease in cholesterol content in themicrodomain in where cholesterol is enriched.

Ratios of Cholesterol to Gangliosides and Cholesterol to Sphingomyelin.Animals fed the GG diet showed a highly significant reduction in theratio of cholesterol to GG, from 31.3 to 18.9, in intestinalmicrodomains when compared to animals fed the CONT diet (FIG. 7-A).Animals fed the PUFA diet did not exhibit a reduced ratio of cholesterolto ganglioside in intestinal microdomains compared to control animals.Animals fed the GG diet also decreased the ratio of cholesterol to SM inintestinal microdomains. The ratio found was 143, 117 and 63 for animalsfed the CONT, PUFA, and GG diet, respectively (P<0.007; FIG. 7-B). Theseobservations suggest that dietary ganglioside increased SM content morethan GG content in microdomains since the ratio of cholesterol to SM wasmore dramatically reduced by 56% in microdomains while the ratio ofcholesterol to GG was decreased by 40% when compared to control animals.

FIG. 7 shows the ratio of cholesterol/GG (A) and cholesterol/SM (B) inintestinal microdomains after feeding different diets for 2 wks.Intestinal microdomains were prepared by a discontinuous sucrose density(5-35-45%) ultracentrifugation from mucosa homogenates suspended by TMEsolution containing 2% v/v Triton-X 100. GG extracted from the Folchupper phase was purified by Sep-Pak C-18 cartridges and used for colordensitometry analysis of total NANA content at 580 nm (Suzuki 1964). SMfrom the lower phase was identified on ‘H’ TLC plates inchloroform/methanol/2-propanol/0.2% KOH/triethylamine solvent system(45:13.5:37.5:9:27, by vol). Phosphate content in SM was measured by aknown method (Itoh et al., 1986). Cholesterol content was analyzed byusing a test kit. The ratio was obtained by dividing cholesterol contentby either GG (P<0.05) or SM (P<0.01) content (mg/mg protein) in themicrodomain. Data were presented as Mean±SD with n=8 animals in eachgroup.

Caveolin Content in Microdomains. Animals fed the GG diet or the PUFAdiet exhibited significantly lower expression of caveolin protein inintestinal microdomains compared to animals fed the CONT diet (FIG.8-A). The blot intensity of caveolin in animals fed the GG and the PUFAdiet was 55% and 45% lower, respectively, than that observed for animalsfed the CONT diet (FIG. 8-B).

FIG. 8 shows caveolin content determined by western blotting (A), andthe intensity of blots (B) in intestinal microdomains fed control dietor treatment diets for 2 wks. Intestinal microdomains were prepared by adiscontinuous sucrose density (5-35-45%) ultracentrifugation from mucosahomogenates suspended by TME solution containing 2% v/v Triton-X 100.Proteins lysed (approximately 25 mg/lane) in SDS reducing sample bufferloaded onto 15% SDS-PAGE minigels and immunoblotted with anti-caveolin-1antibody. A:lane 1, caveolin standard (21-24 kDa); lane 2-5, Controldiet; lane 6-9; PUFA diet; lane 10-13, GG diet. B: Blot intensity wasanalysed by an Imaging Densitometer and each data represents the Mean±SDderived from five animals (n=5) fed different diets.

Content of Inflammatory Mediators, Diglyceride (DG) and PlateletActivating Factor(PAF). DG and PAF content in microdomains was measuredto determine if dietary ganglioside has potential anti-inflammatoryeffects resulting from reduction of pro-inflammatory signals. Animalsfed either the GG diet or PUFA diet showed lower levels of 1,2-DG andtotal DG content, but not in 1,3-DG, in microdomains compared to controlanimals (Table 7). Animals fed the GG diet significantly decreased1,2-DG and total DG by 44% and 43% of controls, respectively. Animalsfed the PUFA diet exhibited a smaller reduction compared to controlanimals (33% and 32% for 1,2-DG and total DG, respectively). Animals fedeither the GG diet or the PUFA diet showed significantly lower levels ofPAF in microdomains when compared to the animals fed the CONT diet (FIG.6-B). Feeding the GG or the PUFA diet decreased PAF content up to 59%and 47%, respectively. Taken together, these results suggest thatdietary ganglioside and PUFA have potential anti-inflammatory effects indeveloping animals and that dietary ganglioside is more effective inreducing two inflammatory factors than feeding PUFA.

TABLE 7 Content of 1,2-, 1,3-, and Total Diglyceride in Rat IntestinalMicrodomains Fed Either Control or Experimental Diets¹ Diet TreatmentControl PUFA GG 1,2-diglyceride² 15.4 ± 2.3^(a) 10.4 ± 3.3^(b) 8.6 ±4.4^(b) 1,3-diglyceride  1.7 ± 0.2  1.3 ± 0.6 1.2 ± 0.2 Totaldiglyceride 17.1 ± 2.2^(a) 11.7 ± 3.8^(b) 9.8 ± 4.6^(b) ¹Values aremeans ± SD of 8 rats. Within a row, values with different superscriptletters are significantly different at P < 0.009. ²Content was expressedas ug/mg protein.

Discussion

Diet Induced-Change. Diet induced-change in the microdomain GD3 and GM3Content is significant because GD3 and GM3 are involved in cellularfunction. GM3 is co-localized with signalling molecules such as c-Src,Rho, and Fak in microdomains (Iwabuchi et al., 2000; Yamamura et al.,1997). GD3 activates T-cells (Ortaldo et al, 1996) and has ananticarcinogenic effect in the mouse colon (Schmelz et al., 2000) Thus,these results may suggest that decreased GM3 alters signals related tothese molecules and that increased GD3 (FIG. 6-(A)) enhances immunefunction and gut protection during development. Our results showingaccumulation of dietary gangliosides in the microdomains are supportedby a previous study demonstrating that administration of [³H]GM3 toNeuro 2a cells showed enrichment of [³H]GM3 in microdomains (Prinetti etal., 1999). These observations suggest that exogenous supplementation ofgangliosides directly incorporates into the microdomain.

Cholesterol is an important lipid involved in compartmentalizingmicrodomains with lipids and proteins (Incardona et al., 2000). Recentstudies found that viral pathogens and cholera toxin appear to reach theER by caveolae (Lencer et al., 1995; Majoul et al., 1996). However,cholesterol reduction in cell membranes inhibits the invasion of HIV-1(Popik et al., 2002), cholera toxin (Wolf et al., 2002), and malarialparasite (Samuel et al., 2001) by disruption of microdomain structure.Cholesterol depletion by drugs down-regulates caveolin gene expression(Hailstones et al., 1998). The present study confirmed that diet-inducedcholesterol reduction in the microdomain also decreased caveolin proteinexpression. The present data therefore suggests a potentialanti-infective effect of dietary ganglioside by reducing cholesterolcontent leading to less caveolin expression.

The present study provides new information indicating dietaryganglioside decreased pro-inflammatory DG and PAF signals in intestinalmicrodomains in developing animals. DG located in microdomains modulatesthe structure and function of microdomains through PKC (Liu et al.,1995; Smart et al., 1995). PAF binds to a PAF receptor in cell membranesto initiate inflammatory signalling events (Flickinger et al., 1999).Together, these observations suggest that PAF may colocalize inmicrodomains as either ether phospholipids or with its receptors andthat diet-induced increase in gangliosides or decrease in cholesterol orcaveolin in the microdomain may disrupt PAF and DG localization in themicrodomain, thereby resulting in inhibition of inflammatory signallingevents.

This example shows that a physiological level of dietary ganglioside hasanti-inflammatory effects in developing animals. Thus present resultsalso illustrate that gangliosides have a protective role in gutdevelopment in infants.

EXAMPLE 5 Dietary Gangliosides and Long-Chain PUFA Alter GD3 andPhospholipids in Neonatal Rat Retina

Dietary long-chain polyunsaturated fatty acids (LCP) such as arachidonicacid (AA) and docosahexaenoic acid (DHA) have been shown to improvevisual acuity in infants (Birch et al., 1998; Carlson et al., 1996;Faldella et al., 1996; and Hoffman et al.,2003). It is thought thatdietary LCP stimulate neonatal retinal development by altering membranephospholipids, which in turn affect cell signaling pathways (Giusto etal., 1997; Huster et al., 2000). During early development, theganglioside composition of the retina also changes significantly wherebyGD3 becomes the primary ganglioside in mammalian retina (Daniotti etal., 1994; Daniotti et al., 1990). Since gangliosides play an importantrole in neuronal cell differentiation and proliferation (Byrne et al.,1983; Fujito et al., 1985; and Ledeenet al., 1998), this change inganglioside profile may indicate retinal maturation. Here we show that aganglioside diet enriched in GD3 increases ganglioside content by 39% inneonatal rat retina, with a relative increase in GD3. Furthermore, wedemonstrate that dietary AA and DHA significantly increase the relativelevels of GD3 in the retina of neonatal rats, providing evidence thatdietary LCP affects ganglioside metabolism in the developing retina andsuggesting a new mechanism by which these dietary lipids may promotematuration of photoreceptor cells.

Introduction

An unusual simplified ganglioside composition is observed in adultretinal photoreceptor cells, compared to that in other central nervoussystem-derived neurons (Dreyfus et al., 1996). Changes in specificganglioside content occurs within photoreceptor cells during postnatalmaturation to reach an end stage characterized by a predominance of GD3in the outer retina and only trace amounts of less complex gangliosides(Dreyfus et al., 1996). Since GD3 is the most prevalent ganglioside infully mature mammalian retinas (Daniotti et al., 1990; Dreyfus et al.,1996), it can be used as a biological marker to evaluate the stage ofretinal development. Although the majority of gangliosides are localizedin the inner retinal membranes, GD3 is primarily found in photoreceptorsin the outer retina (Dreyfus et al., 1996; Dreyfus et al., 1997), whereit plays an important role increasing membrane permeability and fluidity(Barbour et al., 1992; Seyfried et al., 1985).

In embryonic chicken retina, total gangliosides increase up tothree-fold from the 8-day-old embryo to the 15-day-old stage while GD3content decreases by 50% Daniotti et al., 1994). In rats, GM1 isdownregulated and GD1b is upregulated during the period corresponding toformation of the outer segment, a time which correlates with onset ofretinal function (Fontaine et al., 1998). The outer segments,photoreceptor cells, synaptic cells and rhodopsin kinase in the ratretina become functionally active between 10 and 30 days after birth(Fontaine et al., 1998; Ho et al., 1986) during which time GD3 becomesthe predominant ganglioside (Daniotti et al., 1990; Dreyfus et al.,1996; Dreyfus et al., 1997).

Dietary long-chain polyunsaturated fatty acids (LCP) such asdocosahexaenoic acid (DHA) and arachidonic acid (AA) influence the lipidcomposition of retinal membranes, particularly during developmentalstages (Birch et al., 1998; Carlson et al., 1996; Faldella et al., 1996;Anderson et al., 1976; Carrie et al., 2002; and Suh et al., 1994).

Dietary DHA alters the lipid composition of neuronal tissues in retinaand brain, affecting the turnover time for rhodopsin in photoreceptormembranes (Carrie et al., 2002; and Suh et al., 1994). Dietary DHAincreases DHA levels, and levels of other very long-chain fatty acids inrod outer segment membranes in young rats, whereas it does not changethe fatty acid composition of these retinal membranes in mature rats(Nishizawa et al., 2003; Xi et al., 2003). Inclusion of DHA and AA ininfant formulas improves visual development and acuity in infants, butlittle is known about the biological basis for this effect. Gangliosidesare associated with neuronal cell differentiation and proliferationprocesses including migration, neurite outgrowth, axon generation, andsynapse formation (Byrne et al., 1983; Fujito 1985; Ledeen et al., 1998;and Mendez-Otero et al., 2003), processes crucial to visual maturation,but the influence of dietary lipids on retinal ganglioside compositionduring development is poorly understood.

In this example, we hypothesized that dietary gangliosides and LCP couldexert biological effects on visual development through modification ofretinal gangliosides, which change in composition during maturation. Totest this hypothesis, we devised a study that would explore whether anLCP-enriched diet (LCP diet) or a ganglioside-enriched diet (GG diet)could alter retinal ganglioside composition during early development ofneonatal rats. We found that the GG diet caused a 39% increase in thetotal retinal ganglioside content, indicating that gangliosides can beabsorbed and incorporated into body tissues (FIG. 9).

More unusual was the finding that the LCP diet as well as the GG dietcaused significant increases in the relative proportion of GD3, by 19%and 13%, respectively (Table 8). The specific increase in thisparticular ganglioside may indicate that dietary ganglioside or LCPstimulate neuronal development in neonates through enhancing theexpression of GD3, with possible implications for neonatal developmentof visual neural pathways and photoreceptor cell function.

Concurrent with changes in ganglioside profile was an alteration inretinal phospholipid composition attributed to both the GG and the LCPdiets. Both diets were associated with increases in the relative amountsof phosphatidylinositol and lyso-phosphatidylethanolamine, and adecrease in phosphatidylserine and phosphatidylcholine (Table 9). Therewas no effect of the GG diet on total phospholipids, whereas the LCPdiet was associated with a decrease in total retinal phospholipids (FIG.9). All other parameters measured, including total retinal cholesterolcontent, remained unchanged with diet treatment. Phospholipid turnoveralters electric surface potential by affecting calcium and cationconcentration in retinal rod outer segments (Huster et al., 2000) and istightly regulated by light and phosphorylation-dephosphorylationreactions (Giusto et al., 1997). Thus, compositional changes in retinalphospholipids in response to dietary ganglioside or LCP may affect lightadaptation and activation of protein kinases, which ultimately may leadto enhanced development of retinal function in neonates.

Our study demonstrates that even small physiologic amounts of dietarygangliosides or LCP can have profound effects on the lipid profile ofmembranes within the developing retina. Further study is needed toassess the mechanism by which these dietary lipids exert the observedeffects on retinal lipid composition and to determine whether otherneural tissues are similarly affected.

Materials and Methods

Animals and Diets. Two experimental diets or a control diet were fed tomale weanling (18-day-old, 40+4.5 g) Sprague-Dawley rats for two weeks.Animals had free access to water and one of three semi-purified dietscontaining 20% (w/w) fat. The control diet was formulated as 80% basaldiet 25 plus 20% fat as a triglyceride blend, reflecting the overall fatcomposition of infant formula. The LCP-enriched diet (LCP diet) wasformulated by including 1% AA and 0.5% DHA by weight as triglyceride inthe control diet. The ganglioside-enriched diet (GG diet) was formulatedby including in the control diet 0.1% by weight of aganglioside-enriched powder (70-80% GD3, 9% GD lb, 5% GM3, 6% othergangliosides). The GG diet also contained 0.2% (w/w) phospholipid and0.07% (w/w) cholesterol. Body weight and food intake were measured everyother day throughout the experimental period.

Collection of Retina, Lipid Extraction and Ganglioside Separation. Afterdecapitation of animals, whole retinas were removed. All samples wereweighed and kept in a −70° C. freezer until analysis. Total lipids wereextracted using the Folch method (Folch et al., 1957). Gangliosides wereextracted into the Folch upper phase solution. The lower organic phasewas washed twice with chloroform/methanol/water (3/48/47, v/v/v) and theupper phase extracts combined. Gangliosides were purified by passing theupper phase extract through Sep-Pak™ C18 cartridges (Waters Corporation,Milford, Mass.) preconditioned with 10 mL of methanol, 20 mL ofchloroform/methanol (2/1, v/v), and 10 mL of methanol. Cartridges werethen washed with 20 mL of distilled water to remove salts andwater-soluble contaminants. Gangliosides were eluted with 5 mL ofmethanol and 20 mL of chloroform/methanol (2/1, v/v), dried under N₂ gasand then redissolved with 500 mL of chloroform/methanol (2/1, v/v).Gangliosides were stored at −70° C. until analysis.

Analysis of Ganglioside Content. Measurement of total gangliosides asN-acetyl neuraminic acid (NANA) was performed as described by Suzuki(1964). An aliquot of the purified ganglioside sample was dried under N₂gas and dissolved with 0.5 mL each of distilled H₂O and resorcinol-HCl(Svennerholm, 1957). The purple-blue color developed by heating wasextracted into butyl acetate/butanol (85/15, v/v). Optical density wasread at 580 nm. Commercial NANA (Sigma, Mo.) was used as a standard.

Individual gangliosides were separated by silica gel high performancethin layer chromatography (HPTLC; Whatman Inc, Clifton, N.J.) in asolvent system of chloroform/methanol/0.2% (w/v) CaCl₂.2H₂O (55/45/10,v/v/v) and identified using ganglioside standards GM3, GM2, GD3 andbovine brain ganglioside mixture (Alexis, San Diego, Calif.). Individualgangliosides were recovered and measured as described above.

Analysis of Phospholipid Content. Phospholipids were separated fromtotal retinal lipids by thin layer chromatography (TLC) on silica gel‘G’ (Fisher Scientific, Calif.) using chloroform/methanol/water,(65:35:6, by vol.). Individual phospholipids were resolved on silica gel‘H’ TLC plates using chloroform/methanol/2-propanol/0.2%KOH/triethylamine (45:13.5:37.5:9:27, by vol). and identified bycomparison to authentic phospholipid standards (Sigma, Mo.). Plates werevisualized by 0.1% anilinonaphthalene sulfonic acid under UV exposure.Lipid fractions were recovered and lipid phosphate was measuredaccording to the method of Itoh et al., (1986).

Statistical analysis. Six retinas from three animals were pooled toconstitute one replicate to analyze retinal lipids because of the smallamount of lipids in the retina. Values presented are mean±standarddeviation (SD) from 6 replicates (n=6) except for individualphospholipid analysis (n=5). Significant differences between the controlgroup and experimental groups were determined by one-way analysis ofvariance (ANOVA) with SAS (SAS Institute Inc, V6, Fourth Edition, Cary,N.C.). Significant effects of diet treatment were determined by a Duncanmultiple range test at a significant level of P<0.05.

Results

Animal Growth and Tissues. The initial and final body weight of animals,food consumption or retina weight after 2 weeks feeding of experimentaldiets was not different among groups.

Ganglioside Content, Composition and Ratio of GT1b to GD3 in Retina.Animals fed the GG diet increased total ganglioside content in theretina by 39% (P<0.001) when compared to animals fed the CONT diet,indicating bioavailability of dietary ganglioside. Feeding animalseither the LCP or the GG diet showed an increase in the relativepercentage of GD3 in the retina in comparison with feeding animals theCONT diet (by 19% and 13%, respectively). The composition of GM3, GM1,GD1a, GD1b and GT1b was not changed by either diet fed. The ratio ofGT1b to GD3 in the retina was reduced in the both LCP and GG fed animalscompared to animals fed the CONT diet.

FIG. 9 illustrates the total content of a) gangliosides and b)phospholipids in the retina of control and treatment groups. Values arethe mean±SD of 6 samples. Asterisks represent significant difference atlevels of p<0.0008 and p<0.001, respectively.

TABLE 8 Percent composition of gangliosides in the retina of animals feddifferent diets¹ Significant effect Ganglioside² Control LCP GG of diet(p < x) GM3  7.6 ± 2.8  5.8 ± 3.2  7.7 ± 2.3 GM1  7.3 ± 1.8  6.8 ± 2.0 7.7 ± 2.2 GD3  25.0 ± 1.8^(b)  29.8 ± 1.7^(a)  28.2 ± 35^(a) 0.01 GD1a14.3 ± 3.3 15.4 ± 3.5 16.1 ± 1.8 GD1b 19.1 ± 2.2 19.0 ± 2.4 16.7 ± 1.0GT1b³ 26.9 ± 2.9 23.2 ± 3.1 23.6 ± 1.3 ¹Values are mean ± SD where n = 6for each group. Percentage of gangliosides was measured as % of totalNANA content (μg/retina) in the ganglioside fraction. ²Nomenclature ofgangliosides is described by Svennerholm. ³GT1b fraction included GQ1bfraction because of close proximity during TLC separation.

Phospholipid Composition. Feeding animals the LCP diet, but not the GGdiet, reduced total phospholipid content in the retina compared toanimals fed the CONT diet. Animals fed either the LCP diet or the GGdiet showed lower levels of phosphatidylinositol andlyso-phosphatidylethanolamine (PE) and higher levels ofphosphatidylserine and phosphatidylcholine (PC) compared to animals fedthe CONT diet. PE and sphingomyelin (SM) were not changed by either diettreatment fed. Ratios of PE to PC, major phospholipids in the retina,and PC to SM were not affected by both the LCP and GG diet treatment.

TABLE 9 Percent composition of phospholipids in the retina of animalsfed different diets¹ Significant effect of Phospholipid² Control LCP GGdiet (p < x) PE 35.2 ± 4.7 33.4 ± 1.5 34.4 ± 2.3 PI  7.4 ± 1.6^(a)   5.1± 1.3^(b)  4.6 ± 0.7^(b) 0.01 PS  2.3 ± 0.4^(b)  2.8 ± 0.4^(a)  3.1 ±0.1^(a) 0.01 LPE  8.3 ± 0.6^(a)  6.6 ± 0.4^(b)  6.4 ± 1.0^(b) 0.001 PC40.4 ± 3.3^(b) 45.5 ± 2.8^(a) 45.6 ± 2.7^(a) 0.04 SM  6.5 ± 1.3  6.6 ±2.2  6.0 ± 0.7 ¹Values are the mean ± SD for n = 5 in each group.Percentage of phospholipids was measured as phosphate content(μg/retina) in the phospholipid fraction. ²PE =phosphatidylethanolamine, PI = phosphatidylinositol, PS =phosphatidylserine, LPE = lysophosphatidylethanolamine, PC =phosphatidylcholine, SM = sphingomyelin

Cholesterol Content. Cholesterol content in animals fed either the LCPor GG diet was not different from that of animals fed the CONT diet.Animals fed dietary gangliosides exhibited a highly significantreduction in the ratio of cholesterol to gangliosides and phospholipidto gangliosides compared to retinas of animals fed the CONT diet, (28%,P<0.005 and 30%, P<0.0003, respectively). Feeding animals the LCP dietreduced the ratio of cholesterol to gangliosides and phospholipids togangliosides, but not the ratio of cholesterol to SM, compared toanimals fed the CONT diet. The ratio of cholesterol to phospholipids inanimals fed the LCP increased compared to animals fed the CONT diet. Nochange was observed in the ratio of cholesterol to phospholipid andcholesterol to SM in animals fed the GG diet compared to controls.

Discussion

Dietary GG or LCP modifies the lipid classes and the composition ofgangliosides and phospholipids in the developing retina. For example,animals fed the LCP diet increased the relative percentage of GD3, butnot total ganglioside content, compared to animals fed the CONT diet.The increase in the relative percentage of GD3 was accompanied withsignificant changes in total and individual phospholipids. The effect ofdietary LCP on the compositional change of GD3 may suggest that dietaryLCP influence activity of GD3 synthase, an enzyme in the outer retinarequired to synthesize GD3 from GM3 (Daniotti et al., 1992). Traffickingof DHA-containing PL from the trans-Golgi network to the retina outersegment is accompanied with rhodopsin (Rodriguez et al., 1997).Sphingolipids including GG are enriched in microdomains called lipidrafts or caveolae, which are important domains for lipid trafficking.Thus, the present study suggests that diet-induced increase in GD3induced by the LCP diet may influence the trafficking of DHA andrhodopsin from the trans-Golgi network to the outer segment of theretina. This result also imply that beneficial effects known thatdietary LCP influence visual acuity by altering the LCP composition inthe retinal membrane may be a synergistic effect of the compositionalchange of gangliosides in the retina.

In the retina of the rat, the outer segments, photoreceptor cells,synaptic cells and rhodopsin kinase become functionally active between10 days and 1 month after birth (Fontaine et al., 1998; Ho et al., 1986)while GD3 becomes the predominant ganglioside (Daniotti et al., 1990;Dreyfus et al., 1997; and Dreyfus et al.,1996). GD3 in the outer retinais involved in increasing membrane permeability and fluidity and isenriched in differentiated retinas. Since animals used in the presentstudy were fed for 2 wks from 17 days of age, this study suggests thatdietary LCP and GG may stimulate retinal maturation and development byincreasing GD3 content.

Change in the ratio of cholesterol to gangliosides may induce signaltransduction for retinal development as known functional involvement oflipid microdomains. The finding that animals fed the LCP or the GG dietshowed significant changes in the ratio of cholesterol to gangliosidessuggests structural and functional changes of microdomains in retinalmembranes. For instance, administration of gangliosides into the plasmamembrane of MDCK cells displaces GPI-anchored signaling proteins frommicrodomains (Simons et al., 1999). Exogenous addition of [³H]GM3 tomouse neuroblastoma Neuro2a cells shows enrichment of [³H]GM3 inmicrodomains resulting in induction of neuritogenesis by c-SRCactivation (Prinetti et al., 1999).

In summary, this study demonstrates that dietary LCP and gangliosidesmodify metabolism of phospholipids and gangliosides in developingretinal membranes. The present study indicates that a small physiologicamount of phospholipids or gangliosides has a profound effects on thelipid profile of membranes in the retina. The bioavailability ofgangliosides in the diet is high rapidly altering the GD3 composition instructural components of the photoreceptor membrane. Dietarygangliosides would thus alter ganglioside content in other neuronal celltypes.

EXAMPLE 6 Diet Induces Change in Membrane Gangliosides in the IntestinalMucosa, Plasma and Brain

In this example the role of gangliosides in plasma membranes ofmammalian cells as biologically important molecules is examined.Gangliosides are involved in cell differentiation, proliferation,neuritogenesis, growth, inhibition, signaling and apoptosis (Byrne etal., 1983; De Maria et al., 1997; Ledeen,1989; and et al.,1996). GM32and GM1 can act as receptors for enterotoxins such as rotavirus inanimals (Rolsma et al., 1998) and Vibrio cholerae and Escherichia coliin humans (Laegreid et al., 1987). GD3 stimulates T-cell activation inhuman peripheral blood lymphocytes (Ortaldo et al., 1996; Welte et al.,1987). Ganglioside content and composition is significantly differentbetween stages of development. For example, in human brain, GM1increases from birth to the age of 1 year while GD1a decreases in thewhite matter (Vanier et al., 1971). The ratio of GM3/GD3 is about0.2-0.3 in human colostrum while the ratio is greater than 3 in maturemilk since GM3 gradually becomes a major ganglioside upon lactation(Takamizawa et al., 1986). Recent studies have also shownneuroprotective effects of GM 1. GM 1 treatment of neonatal ratsprevents hypoxic damage (Krajnc et al., 1994). GM1 intervitreallyinjected is protective against rat retinal ischemia induced by pressure(Mohand-Said et al., 1997), and intravenous administration of GM 1reduces infarct volume caused by focal cerebral ischemia (Lazzaro etal., 1994). Despite evidence that gangliosides are involved indevelopment, it is still not clear if dietary gangliosides inducechanges in membrane gangliosides or where GM3 and GD3 are localized inthe enterocyte membrane. This information is vital to understanding thebiological functions of these molecules during the period of developmentin which their role is the most significant.

The cholesterol content of membrane is also important in maintaining anoptimal cell membrane environment. Recent work shows that cholesterolhomeostasis is related to sphingomyelin content (Slotte 1999), andcholesterol absorption is regulated in part by sphingomyelin content inintestinal cell membranes (Chen et al., 1992). Cholesterol is enrichedin membrane microdomains such as rafts and caveolae, perhaps mediatingsignal transduction (Maekawa et al., 1999). Gangliosides have the sameceramide as the anchored hydrophobic moiety of sphingomyelin, with theonly difference occurring in a polar head group. No studies havereported the effect of gangliosides on cholesterol turnover or membranecontent of cholesterol in vivo.

The present research was designed to determine whether dietaryganglioside increases the content of total and individual gangliosidesand affects the level of cholesterol, thereby changing the ratio ofcholesterol to gangliosides in the intestinal mucosa, plasma and brainin developing rats. This is also the first study showing thelocalization of GM3 and GD3 in the enterocyte membrane.

Materials and Methods

Animals and diets. Male 18-day-old Sprague Dawley rats (n=24), averagebody weight 40±4.5 g, were randomly separated into 3 groups of 8 with 2or 3 rats housed in each polypropylene cage. Animals were maintained ata constant temperature of 23° C. and a 12 h light/dark cycle. Animalshad free access to water and one of three semi-purified diets containing20% (w/w) fat for 2 weeks. The composition of the basal diets fed hasbeen reported (Clandinin et al., 1980). Animal body weight and foodintake were recorded every other day throughout the experiment. Thecontrol diet fat was a blend of triglyceride, which reflected the fatcomposition of an existing infant formula. Dietary fatty acids werecomposed of about 31% saturated fatty acids, 48% monounsaturated fattyacids and 21% polyunsaturated fatty acids with a ratio of 18:2n-6 to18:3n-3 of 7.1. Two experimental diets were formulated by adding eithersphingomyelin (SM, 1% w/w, Sigma, Mo., USA) or a ganglioside-enrichedlipid (GG, 0.1% w/w, New Zealand Dairy, New Zealand) to the controldiet. Ganglioside-enriched lipid consisted of about 45-50% (w/w)phospholipids and 15-20% (w/w) gangliosides. The cholesterol content was<0.35% w/w total lipid. The ganglioside fraction contained about 80% w/wGD3, with GD1b, GM3 and other gangliosides accounting for 9, 5 and 6%w/w, respectively.

Collection of Samples. After anesthetizing animals with halothane, bloodwas collected by cardiac puncture and immediately spun at 1000×g (JA-20Rotor, Beckman, USA) for 30 min to recover plasma. Followingdecapitation, the brain and small intestine (jejunum to ileum) wereexcised. The intestine was washed with ice-cold 0.9% saline solution toremove visible mucus and dietary debris, opened and moisture wascarefully removed with a paper towel to correctly measure mucosa weight.Intestinal mucosa was scraped off with a glass slide on an ice-coldglass plate. All mucosa samples were weighed and kept in a −70° C.freezer until extraction.

Immunofluorescence study. Intestinal sections were collected fromanimals. Samples were washed with cold phosphate buffered saline (4°C.), cut into 5 mm pieces and fixed with 4% paraformaldehyde in PBS for1 h at 4° C. After washing with cold PBS, samples were infiltrated with15% and 30% sucrose in PBS for 90 min and overnight, respectively, at 4°C. for cryostat protection. The tissue sections were placed on plasticmolds and covered with embedding medium by optimal cutting temperature(O.C.T.; Tissue Tek, Sakura Finetek USA) on dry ice. Frozen sections (1mm thickness) were mounted on polylysine-coated microscope slides andwashed in cold PBS for 30 min at room temperature. The sections wereblocked with 2% bovine serum albumin in PBS for 1 h at room temperatureand then incubated with anti-ganglioside GM3 (Mouse IgM, SeikagakuColo., USA) (diluted 1:25), or anti-ganglioside GD3 monoclonal antibody(Mouse IgM, Seikagaku Colo., USA) (diluted 1:25), for 2 h at roomtemperature. After washing the sections 3 times for 10 min with coldPBS, the sections were incubated with fluorescein isothiocyanate(FITC)-conjugated anti-mouse IgM (Sigma, Mo., USA) (diluted 1:300) for 1h at room temperature in the dark room and washed with PBS again 3 timesfor 10 min. After staining, a drop of N-propyl gallate was added ontothe section before mounting a cover slip. All samples were sealed withnail polish and examined with a confocal microscope (Zeiss ConfocalLaser Microscope 510, Carl Zeiss, Germany) with an Argon laser line (488nm excitation, barrier filter LP505, Plan-Neofluar 40X, 1.3 oilimmersion objective).

Ganglioside extraction and purification. Total lipid was extracted usingthe Folch method (Folch et al.,1957). For extracting gangliosides, thelower phase was washed twice with Folch upper phase solution(chloroform/methanol/water, 3/48/47 by vol.). The upper phasegangliosides were pooled and then purified by passing through Sep-Pak™C18 cartridges (Waters Corporation, Milford, Mass., USA) prewashed with10 mL of methanol, 20 mL of chloroform/methanol (2/1, v/v), and 10 mL ofmethanol as described by Williams and McCluer (1980). The upper phaseextract was loaded onto C18 cartridges. Cartridges were then washed with20 mL of distilled water to remove salts and water-soluble contaminants.Gangliosides were eluted with 5 mL of methanol and 20 mL ofchloroform/methanol (2/1, v/v), dried under N₂ gas and then redissolvedwith 500 mL of chloroform/methanol (2/1, v/v). Gangliosides were storedat −70° C. until analysis.

Analysis of total and individual ganglioside content by measuring NANA

Total NANA-gangliosides were measured as described by Suzuki (1964). Analiquot of the ganglioside sample purified by Sep-Pak C 18 cartridgeswas dried under N₂ gas and dissolved with each of 0.5 mL of distilledH2O and resorcinol-HCl (Svennerholm 1957) in screw-capped Teflon-linedtubes. The purple blue color developed by heating was extracted intobutylacetate/butanol (85/15, v/v) solvent. Optical density was read by aspectrophotometer (Hewlett Packard, 8452A) at 580 nm. For quantitativeanalysis, N-acetyl neuraminic acid (Sigma, Mo., USA) was used as astandard.

Individual gangliosides were separated by silica gel high performancethin layer chromatography (HPTLC; Whatman Inc, Clifton, N.J., USA) alongwith standards of ganglioside GM3, GM2, GD3 and bovine brain gangliosidemixture (Alexis, San Diego, Calif., USA) in a solvent system ofchloroform/methanol/0.2% (w/v) CaCl₂.2H₂O (55/45/10, by vol.).Individual ganglioside fractions were scraped off and measured asdescribed above.

Cholesterol assay. Cholesterol analysis was completed with a test kit(Sigma, Mo., USA).

Statistical analysis. The values shown are means±standard deviation(SD). Significant differences between the control group and experimentalgroups were determined by one-way analysis of variance (ANOVA) with SAS.Significant effects of diet treatment were determined by a Duncanmultiple range test at a significance level of p<0.05.

Results

Animal growth and tissues. There were no significant differences amongthe control, SM and GG groups either in terms of the initial body weightof animals or their final weight after 2 weeks feeding of experimentaldiets (Table 10). Brain weight, intestinal mucosal weight and intestinallength were not affected by dietary treatment. Food consumption was notinfluenced by diet.

TABLE 10 Weight of Animals and Tissues Fed Control or ExperimentalDiets¹ Diet Treatment: Control SM GG Initial Body Wt. (g) 39.9 ± 4.5 40.6 ± 4.4  40.3 ± 4.5  Final Body Wt. (g)  117 ± 13.5  118 ± 12.1  120± 13.1 Intestine Length (cm) 82.0 ± 5.8  79.8 ± 6.2   84 ± 6.0 MucosaWt. (g) 2.1 ± 0.3 2.2 ± 0.3 2.1 ± 0.3 Brain Wt. (g) 1.8 ± 0.1 1.8 ± 0.11.7 ± 0.1 ¹Values are mean ± SD with 8, 8, and 6 for mucosa, plasma andbrain, respectively.

The localization of GM3 and GD3 in the enterocyte by confocalmicroscopy. Localization of GM3 and GD3 in the enterocyte was determinedusing a confocal microscope. GM3 stained with FITC-conjugate was almostexclusively localized at the apical membrane of the enterocyte (FIG.10). The majority of the GD3 was found in the basolateral membrane ofthe enterocyte with only minor staining in the apical membrane (FIG.10).

FIG. 10 shows immunofluorescent detection of GM3 localization.Immunofluorescent detection of GM3 in intestinal villi was analyzed with(B, D) or without (A, C) treatment of anti-GM3 visualized withFITC-conjugate IgM and confocal microscopy. GM3 was almost exclusivelylocalized at the apical membrane of enterocytes.

FIG. 11 shows Immunofluorescent detection of GD3 localization.Immunofluorescent detection of GD3 in intestinal villi was analyzed with(B, D) or without (A, C) treatment of anti-GD3 visualized withFITC-conjugate IgM and confocal microscopy. GD3 was mostly localized atthe basolateral membrane with minor staining at the apical membrane ofenterocytes.

Total ganglioside content in tissues and plasma. The effect of dietaryganglioside on total ganglioside content of the intestinal mucosa,plasma and brain from animals fed the control and experimental diets for2 weeks are shown (FIG. 12). Animals fed the GG diet had significantlyhigher ganglioside content in the intestinal mucosa, plasma and braincompared to control animals. The highest tissue level of ganglioside wasobserved in the intestinal mucosal membrane. The lowest level ofganglioside was found in brain membrane. No change in total gangliosidecontent of either tissues or plasma was found after feeding the SM diet.

FIG. 12 illustrates the effect of dietary treatment on total content ofgangliosides in (A) the intestinal mucosa, (B) plasma and (C) brain foranimals fed either the control or experimental diet for two weeks.Values are means±SD, p<0.02 for A and C, and p<0.003 for B. Treatmentvalues represent the means of n=7, 8 and 6 animals for mucosa, plasmaand brain, respectively.

Individual ganglioside composition in tissues and plasma. Animals fedthe GG diet showed a higher level of GD3 and GQ1b in the intestinalmucosal membrane compared to control animals (Table 11, p<0.001 andp<0.05, respectively). This result was accompanied by significantreduction of ganglioside GM3, which is normally a major ganglioside inthe intestinal mucosa. Feeding the GG diet did not affect the level ofGM2, GM1, GD1a, GD1b or GT1b in the intestinal mucosa compared to thecontrol. Animals fed the SM diet did not exhibit any change inindividual ganglioside patterns, but showed increase in GM2 compared tocontrol animals (p<0.05).

TABLE 11 Composition of Gangliosides in the Rat Intestinal Mucosa FedEither Control or Experimental Diets¹ Control SM GG Diet Treatment: (%)³Ganglioside² GM3 83.5 ± 6.7^(a)  82.4 ± 7.5^(a)  76.4 ± 6.9^(b) GM2 2.0± 0.9^(b) 4.1 ± 2.4^(a)  2.8 ± 0.8^(ab) GM1 2.7 ± 1.7  3.0 ± 1.7   1.7 ±1.2 GD3⁴ 3.2 ± 1.3^(b) 2.2 ± 0.9^(b)  7.5 ± 1.9^(a) GD1a 2.3 ± 1.5  2.8± 1.5   1.9 ± 0.9 GD1b 1.9 ± 1.0  1.5 ± 0.8   2.2 ± 1.4 GT1b 2.1 ± 2.2 1.6 ± 1.6   3.1 ± 2.2 GQ1b 2.3 ± 1.6^(b) 2.6 ± 1.6^(b)  4.5 ± 1.6^(a)¹Values are mean ± SD of 7 rats. Within a row, values with differentsuperscript letters are significantly different at P < 0.05.²Nomenclature was referred from Svennerholm. ³Expressed as a % of totalganglioside fraction. ⁴Values are significantly different at P < 0.001.

In plasma, only four major ganglioside fractions (GM3, GD1a, GD1b andGT1b) were measured since the total ganglioside content was much lowercompared to either tissue. The GD3 fraction could not be quantifiedbecause unknown fraction partially overlapped with GD3. Two minorgangliosides, GM2 and GM 1, were faintly visible on the TLC plate.Animals fed the GG or SM diet did not show a significant change in theindividual ganglioside composition of plasma compared to control animals(Table 12). There was a trend toward increased GM3 in animals fed the GGdiet compared to control animals (p<0.07). GD1a and GD1b represented33.9% to 36.0% and 16.1 to 19.6% of the plasma ganglioside fraction,respectively.

TABLE 12 Composition of Gangliosides in Plasma of Rats Fed EitherControl or Experimental Diets¹ Control SM GG Diet Treatment: (%)²Ganglioside GM3 21.6 ± 3.3 26.5 ± 3.5 28.5 ± 5.3 GD1a 36.0 ± 5.5 30.2 ±3.6 33.9 ± 2.7 GD1b 16.3 ± 1.8 19.6 ± 6.2 16.1 ± 3.5 GT1b 26.1 ± 2.223.7 ± 4.1 21.4 ± 5.5 ¹Values are mean ± SD of 5 samples. ²Expressed asa % of the total ganglioside fraction.

Brain ganglioside fractions were separated into 14 fractions as shown bySonnino et al. (1983). The five major gangliosides were GD1a, GT1b,GD1b, GQ1b and GM1. Minor components were GT1a, GD3 and GM3. Theremaining six fractions were collected as others. Animals fed the GGdiet or SM diet exhibited no change in individual gangliosidecomposition in the brain compared to control animals (Table 13), but thetotal ganglioside content increased.

TABLE 13 Composition of Gangliosides in Brain of Rats Fed Either Controlor Experimental Diets¹ DIET TREATMENT CONTROL SM GANGLIOSIDE (%)² GM32.5 ± 0.8 2.8 ± 1.5 2.6 ± 0.5 GM1 6.3 ± 0.3 6.0 ± 0.3 6.2 ± 0.5 GD3 3.0± 0.7 3.0 ± 0.3 3.0 ± 0.5 GD1a 23.2 ± 2.8  24.0 ± 2.0  23.6 ± 2.5  GT1a4.3 ± 0.4 4.5 ± 0.3 4.8 ± 0.5 GD1b 11.3 ± 0.8  11.0 ± 1.2  11.4 ± 0.9 GT1b 22.4 ± 2.5  21.7 ± 1.6  21.9 ± 0.9  GQ1b 6.8 ± 0.3 6.5 ± 0.1 6.4 ±0.6 Others³ 20.3 ± 4.4  20.5 ± 3.8  20.0 ± 3.4  ¹Values are mean ± SD of4 rats. ²Expressed as a % of the total ganglioside fraction. ³Six minorfractions were gathered as others.

Cholesterol content in tissues and plasma. Animals fed the GG dietshowed a lower level of cholesterol in the intestinal mucosa and brain,but not in the plasma, compared to animals fed the control diet (FIG.13). Animals fed the SM diet did not exhibit any change in totalcholesterol content in the plasma and brain, but exhibited a significantdifference in the intestinal mucosa compared to control animals(p<0.03). Unlike a previous report (Slotte 1999), the SM diet did notincrease cholesterol content in the intestinal membrane, but reducedcholesterol content compared to animals fed the control diet. Animalsfed the GG diet showed lower cholesterol in plasma compared to those fedthe SM diet (FIG. 13B).

FIG. 13 illustrates the effect of dietary treatment on cholesterolcontent in (A) the intestinal mucosa, (B) plasma and (C) brain ofanimals fed either the control or experimental diets for two weeks.Values are means±SD, p<0.03 for A and B, and p<0.0002 for C. Treatmentvalues represent the means of n=7, 8 and 6 animals for mucosa, plasmaand brain, respectively

Ratio of cholesterol to gangliosides. Animals fed the GG diet showed ahighly significant reduction in the ratio of cholesterol to gangliosidein the intestinal mucosa, plasma and brain compared to animals fed thecontrol diet (FIG. 14), and a lower level of cholesterol in plasmacompared to feeding SM, which is an appropriate single lipid control.Animals fed the SM diet also exhibited a reduced ratio of cholesterol toganglioside in the intestinal mucosa and brain, but not in plasmacompared to control animals. Of the three dietary treatments, the lowestratio of cholesterol to ganglioside was observed in animals fed the GGdiet. In contrast, the highest ratio was found in animals fed thecontrol diet in both tissues but not in the plasma. In the plasma, thehighest ratio of cholesterol to gangliosides was observed in animals fedthe SM diet.

FIG. 14 illustrates the effects of dietary treatment on the ratio ofcholesterol to ganglioside in (A) the intestinal mucosa, (B) plasma and(C) brain of animals fed either the control or experimental diets fortwo weeks. Data values are means±SD, A: p<0.0007, B: p<0.002 and C:p<0.0001. Treatment values represent the means of n=7, 8 and 6 animalsfor mucosa, plasma and brain, respectively. The ratio was obtained bydividing the tissue cholesterol content (mg/g wet weight) by the totaltissue content of ganglioside (mg/g wet weight).

Discussion

The notion that gangliosides may have beneficial effects in developmenthas prompted studies of the influence of dietary ganglioside onintestinal and brain development. GM1 acts as a receptor for choleratoxin and E. coli. GM3 is the major ganglioside in the enterocyte ofhumans and animals (Holgersson et al., 1988; Bouhours et al., 1983) butthe intracellular localization is not known. The present study clearlyshows that gangliosides GM3 and GD3 are localized at the apical andbasolateral membrane of the enterocyte, respectively. As GM3 is themajor ganglioside in the enterocyte of humans and animals, the presentstudy suggests that the different localizations of GM3 and GD3 probablyhave different biologic and/or physiologic functions for protection anddevelopment. GM1 bound with cholera toxin is transcytosized from theapical to the basolateral membrane to activate the basolateral effecter,adenylate cyclase (Lencer et al., 1995). The present study did notexamine the possibility that GM3, like GM1, may also be transcytosized.Total ganglioside content and individual ganglioside composition weresignificantly changed, but the degree of change could not bequantitatively estimated by confocal microscopy.

Dietary ganglioside significantly increases membrane ganglioside contentin the intestinal mucosa, plasma and brain, thereby having potential tocause developmental change. Increased membrane ganglioside in theintestinal mucosa might influence enterocyte immune functions sincegangliosides activate immune functions and provide attachment sites forenterotoxins and viruses. Neonatal intestinal mucosa has a relativelylow level of immunoglobulin-containing cells after birth to about 2weeks of age (Perkkio et al., 1980). Mother's milk and the intestinehave a compensatory high level of gangliosides during this period(Bouhours et al., 1983; Carlson 1985), suggesting that gangliosides mayhave a key role in protection of the neonate from antigens.

In the present example, increased membrane ganglioside was accompaniedby changes in the individual ganglioside composition of the intestinalmucosa. GD3 was increased by feeding gangliosides while the majorganglioside in the intestine, GM3, decreased compared to controlanimals. Since GD3 activates T-cells and has an anticarcinogenic effectin the mouse colon (Schmelz et al., 2000), it is logical to suggest thatincreased GD3 might influence enterocyte functions and infection byaltering the interaction with the developing immune system. In plasma,diet treatment considerably increased total gangliosides, but no changewas found in the composition of individual gangliosides. In contrast tohuman serum, where GM3 is the major ganglioside (Senn et al., 1989),GD1a was the major ganglioside in rat plasma. Dietary gangliosidemarkedly increased total ganglioside content in the brain compared toanimals fed the control diet. The present data suggests that dietaryganglioside may affect brain development since an increase ofganglioside content in the brain may effect protection against neuronalinjury, induce neurite growth, and is observed in well-fed animalscompared to undernourished animals (Karlsson et al., 1978; Morgan etal., 1980). The present study agrees with previous results describingthe pattern of major brain gangliosides. In rodents, GM1 increases fromthe 3rd to 24th month (Aydin et al., 2000) during development (Sun etal., 1972). The low level of GM1 observed in the present study may bedue to the younger animal age compared to that of previous work. Thelack of significant change in individual ganglioside patterns observedin the brain may be attributed to a short experimental period (2 weeks),the lack of change in ganglioside patterns in the plasma or specificcontrol of individual ganglioside composition in the brain.

The SM content used in this experiment was relatively much higher (>10fold) than the ganglioside content of the GG diet. This higher level ofdietary SM did not alter tissue ganglioside content, suggesting thatdietary GG is a better source for enhancing membrane GG and that dietarySM may not be used for GG synthesis during the early stage ofdevelopment.

The present example indicates that animals fed the GG diet exhibited asignificant reduction of cholesterol content in the intestinal mucosacompared to animals fed the control diet. A disruption in microdomainstructures caused by reduced cholesterol content may prevent endocytosisof toxins or invasions by bacteria (Parpal et al., 2001; Samuel et al.,2001). A similar result was also observed for animals fed the SM diet.Long term feeding of 1% sphingolipid in the diet significantly reducesplasma cholesterol content (Kobayashi et al., 1997). Our study, inagreement with Imaizumi (Imaizumi et al., 1992), showed that feeding SMfor two weeks did not alter plasma cholesterol content. This observationmay be due to differences in age, diet, species or in vivo and in vitroexperimental conditions.

Animals fed the GG diet showed a significant decrease in cholesterol inthe brain compared to animals fed the control diet. Cholesterol ismaintained in the brain by regulating its de novo synthesis and theuptake of LDL-cholesterol as well as the release of HDL-cholesterol(Bastiaanse et al., 1997). Cholesterol turnover takes place very slowlyin brain (Andersson et al., 1990). In comparison with Kobayashi et al.,(1997), our data suggest that the effect of gangliosides on cholesterolreduction in the brain may be dependent on NANA combined withglycosphingolipid since sphingolipids, such as cerebroside and SM thatdo not contain NANA, do not affect brain cholesterol content.

It appears that individual gangliosides have different roles in theregulation of cell behaviour, as each ganglioside is localized indifferent enterocyte membrane sites. Cholesterol is an important factorinvolved in cell permeability, fluidity (Rietveld et al., 1998), gapjunctions (Malewicz et al., 1990) and membrane microdomains called raftsor caveolae (Brown et al., 1998). Change in membrane cholesterol contentin the intestinal mucosa and brain might affect membrane functionsduring development.

The ratio of cholesterol to gangliosides was decreased in the intestinalmucosa, plasma and brain by feeding the GG diet compared to animals fedthe control diet. Changes in this ratio, in both tissues and plasma,could suggest that dietary gangliosides alter membrane functions. Thissuggestion is supported by early studies showing that changes inmembrane lipid composition (Clandinin et al., 1991) and membranecholesterol content (Rietveld et al., 1998) influence membranefunctions. The present research also suggests that dietary gangliosidesmight affect the traffic of lipids and proteins in membrane microdomainssince GM3 and GD3 are localized in different sites and(glyco)sphingolipids including gangliosides and cholesterol are the mostabundant lipids present in rafts and caveolae (Incardona et al., 2000;Parton, 1994). The functions of caveolae are closely involved withcholesterol content (Incardona et al., 2000). Depletion of cholesterolcontent in caveolae inhibited the MAP kinase complex, stimulated Erkenzymes and increased mitogenesis (Furuchi et al., 1998).

Gene expression of caveolin, a marker of protein for caveolae, wasupregulated with cholesterol (Fielding et al., 1997), suggesting thatcholesterol-protein interaction directly modulates gene expressionimportant for cell development and behaviour. In the sarcolemma,reduction in cholesterol content results in increased Ca²⁺/Mg²⁺-ATPaseactivity and decreased Ca2+/Mg²⁺-ATPase activity when the level ofcholesterol is high (Ortega et al., 1984). It is also logical to suggestthat dietary ganglioside might influence intestinal immune functions bymodulating the lipid profile in membrane lipid rafts since activation ofsignal transduction by IgE receptors and T-cell receptors is dependenton membrane lipid rafts (Moran et al., 1998; Stauffer et al., 1997).

In summary, this study suggests that dietary gangliosides are absorbedby the intestine, remodelled in the enterocyte and induce changes inmembrane total content of ganglioside and cholesterol in the intestinalmucosa and brain. The observations suggest that dietary ganglioside fedat a physiological level will alter membrane lipid profiles thatinfluence membrane functions involved in a wide variety of cellfunctions in neonatal development. Infant formulas have lower levels ofgangliosides and a different ganglioside composition compared to that ofhuman breast milk (Pan et al., 2000; Sanchez-Diaz et al., 1997). Thebioavailability of dietary gangliosides demonstrated in this paper andthe impact on the lipid composition of developing tissues indicates thatthese differences in feeding regimens are of biological importance.

EXAMPLE 7 Dietary Ganglioside: Functions in the Intestine duringDevelopment

This example illustrates the role of dietary gangliosides in decreasinginflammatory factors PAF and DG in microdomains of the rat intestine.The intestines of rats fed either a control diet (Cont) or a diet highin gangliosides (GG), as described in previous Examples, were assessedfor GM3 and GD3 in microdomains. The methodology used for assessingganglioside composition is as described in previous Examples.

The increase in GD3 composition of microdomains for animals fed theganglioside-enriched diet correlated to a decrease in both PAF and DG inmicrodomains. These parameters are indicative of a decrease ininflammatory factors in the intestine and thus show inflammationmediation induced by dietary gangliosides.

FIG. 15 illustrates the composition of GM3 and GD3 in microdomains ofrat intestine. While GM3 is reduced on the GG diet, GD3 is increased.

FIG. 16 illustrates the composition of PAF and DG in microdomains of ratintestine. Both PAF and DG are reduced with a GG diet. Data shown ispresented on a μg/mg protein basis.

FIG. 17 illustrates the caveolin content of microdomains for animals feda control, PUFA or GG diet. The presence of caveolin is reduced for boththe PUFA and GG diets, relative to control. This decrease in caveolinprotein, a marker of microdomains, was observed in rat intestines fromanimals fed a ganglioside-enriched diet. A reduction in caveolin is alsoindicative of a reduced likelihood of bacterial and/or viral infectionthrough an intestinal entry route.

EXAMPLE 8 Dietary Gangliosides Effect Plasma Lipid Content and Ratios ofPlasma Lipids

This example illustrates the effect of dietary gangliosides ondecreasing plasma lipid content, specifically cholesterol andtriglyceride, as well as the lipid ratios cholesterol: NANA andcholesterol: phosphorus in the rat.

Plasma was sampled from rats fed either a Control, SM or GG (high inganglioside) diet, as described in previous Examples. Plasma NANA,phosphorus, cholesterol, and triglyceride, was evaluated as described inprevious Examples.

FIG. 18 illustrates that feeding a diet high in ganglioside resulted indecreased plasma cholesterol and triglyceride. This finding suggestreduced cholesterol and lipid absorption from the intestine. Further,because of the decreased cholesterol and increased NANA with the GGdiet, a striking reduction in the cholesterol: NANA ratio was observed,illustrating that the GG diet effects lipid composition in plasma.

EXAMPLE 9 Preparation of a Crude Fraction Enriched in NaturalGangliosides

This example provides a formulation of natural gangliosides which may beused according to an embodiment of the invention. The crude fractiondescribed is derived from whole milk, and in particular, fat globulesfrom whole milk.

The initial step in preparing the crude fraction involved in MFGMisolation involves separating the fat globules from fresh, uncooledmilk. Centrifugation at low g forces readily separates all but thesmallest fat globules from milk, and helps to minimize physical damageto the fat globules (Patton et al., 1975; Mather, 1987). The second stepin the process is to wash the fat globules, this will remove entrainedor adsorbed components of milk serum as well as caseins and whey (Keenanet al., 1988). Washing involves resuspension and reflotation of lipidglobules in buffered or unbuffered water made isotonic with milk serumby addition of sucrose or sodium chloride (Keenan et al., 1988). Twowash cycles at temperatures above 25° C. is sufficient to remove caseinsand whey proteins from globules prepared in a laboratory centrifuge,while milk separated via a mechanical cream separator may require 3 or 4washes (Keenan et al., 1988). The final composition of the material thatis recovered as MFGM will vary according to the method and extent ofwashing of lipid globules (Keenan et al., 1995), and thus intermittentanalytical testing carried out to ensure that the product yield andcomposition conform to specifications.

The final steps in the procedure release the membrane from the globulevia slow and successive freeze/thaw cycles. Finally, the membrane ispelleted via centrifugation at 100 000×g for 60-90 mins (Patton et al.,1975).

Small milk batches of 10 L can be used to test the method during theinitial phases of progressing to 100 L, and finally to 1000 L batches.

EXAMPLE 10 Gangliosides in Prevention and Treatment of NecrotizingEnterocolitis

Necrotizing enterocolitis is an inflammatory bowel disease of neonatesand remains the leading gastrointestinal emergency in premature infants.Since current treatments disrupt the natural microflora, are invasiveand do not target the specific processes underlying development ofnecrotizing enterocolitis, it remains the leading cause of morbidity andmortality in neonatal intensive care units. Prematurity,hypoxia-ischemia, infection and formula feeding are established riskfactors and breastfeeding is protective.

The role of local vasoactive and inflammatory mediators in theunderlying pathogenesis remains elusive due to limitations in currentmodels. Existing models of necrotizing enterocolitis are limited in thatthey often use animals, immortalized cell lines or diseased tissue tostudy single risk factors. These models do not contain the aspect ofprematurity that is the predisposing factor in infant necrotizingenterocolitis. Moreover, past studies have focused their measurements onplasma or serum vasoactive and inflammatory mediators which reflect thesystemic response rather than the local intestinal response. Severalvasoactive and inflammatory mediators including endothelin-1, nitricoxide, platelet activating factor, leukotrienes, prostaglandins,cytokines (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β),interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10),interleukin-12 (IL-12) and interleukin-18 (IL-18) ) and reactive oxygenspecies may play a synergistic and central role in the inflammatorypathway leading to necrotizing enterocolitis. Serotonin promotesbacteria translocation and is elevated during bowel inflammation, bowelobstruction and infection, but the role of serotonin in necrotizingenterocolitis has not been investigated.

Evidence suggesting that breast-fed infants are one tenth less likely todevelop necrotizing enterocolitis than formula-fed infants leads to thehypothesis that bioactive factors in human milk might be responsible forprotective effects. Gangliosides are sialic acid-containingglycosphingolipids located at the surface of cell membranes and withinthe milk fat globule membrane.

In this example, a model to study the role of gangliosides in theinflammatory response of human neonatal bowel to infection and hypoxiais employed. These data show that gangliosides reduce neonatal bowelnecrosis by reducing production of nitric oxide, LTB4, IL-1b, IL-6 andIL-8 in response to enterotoxic E coli LPS and hypoxia. These findingsreveal that gangliosides improve bowel survival in necrotizingenterocolitis by suppressing the local inflammatory response of humanneonatal bowel to both infection and hypoxia.

The above-described embodiments of the present invention are intended tobe examples only. Alterations, modifications and variations may beeffected to the particular embodiments by those of skill in the artwithout departing from the scope of the invention, which is definedsolely by the claims appended hereto. All documents are hereinincorporated by reference.

1. A method for treating necrotizing enterocolitis in a subject in needthereof, the method comprising administering a formulation comprising atleast one ganglioside to the subject, wherein the formulation comprisesat least 50% GD3 by weight of total ganglioside content.
 2. The methodof claim 1 wherein administering comprises oral consumption.
 3. Themethod according to claim 1, wherein the formulation additionallycomprises a ganglioside selected from the group consisting of: GM1, GM2,GM3, and GD1b.
 4. The method of claim 2 wherein the formulationcomprises a ganglioside supplemented liquid or food.
 5. The method ofclaim 4 wherein said supplemented liquid or food comprises an infantformula or infant food.